Immunology Letters, 41 (1994) 195-199 Elsevier Science B.V.

IMLET 2147

T-cell recognition of an allogeneic RT1-Db u class II MHC peptide

Gerard Murphy *, Rosemarie Dalchau, Kay E. Parker 1, Greta J. Sawyer, Clare A. Carter and John W. Fabre

Division of Cell and Molecular Biology, Institute of Child Health, University of London, 30 Guilford Street, London WCIN 1EH, UK

(Received 25 March 1994; revised 29 April 1994; accepted 2 May 1994)

Key words: Transplantation; Major histocompatibility complex; Processing; Presentation; Indirect allo-recognition

1. Summary

The allo-antibody response of several rat strains to an unconjugated synthetic 20 amino acid peptide de- rived from the c~ helical region of the RT1-D u fl chain was tested. The LEW (RT1 l) and WAG (RT1 u) strains produced little or no antibody; the PVG (RT1 c) and DA (RT1 avl) strains produced moderate amounts of anti- body; while the BN (RT1 n) strain produced strong primary and secondary antibody responses. This sug- gested that the BN strain was able to process and present the RT1-Db u peptide on its class II molecules. In vitro proliferation studies demonstrated that LEW T cells did not respond to the peptide, whereas BN T cells responded strongly, and that the response in the BN strain was found only in the CD4 + T-cell subset. However, immunisation of BN rats with the RT1-Db u peptide failed to cause any acceleration of rejection of WAG skin or kidney grafts. Moreover, BN rats primed with WAG skin and kidney grafts did not produce T cells reactive to the RT1-Db u synthetic peptide. This suggests that the T-cell response of the BN strain to the synthetic major histocompatibility complex peptide was not relevant to the indirect T-cell allo-recognition re- sponse of naturally processed RT1-D u/3 chains.

2. Introduction

The recognition by T cells of allogeneic major histo- compatibility complex (MHC) peptides presented by autologous antigen-presenting cells (APC) (indirect T- cell allo-recognition) was initially demonstrated in vitro

* Corresponding author: G. Murphy, Division of Cell and Molecular Biology, Institute of Child Health, University of London, 30 Guilford Street, London WC1N 1EH, UK. x Current address: Smith Kline Beecham Pharmaceuticals, Harlow.

by defining the fine specificity of T-cell clones [1-3] and in vivo by peptide immunisations [4]. More impor- tantly, T cells responding to donor MHC peptides have been demonstrated to be generated in the course of normal rejection responses [5,6], and indirect T-cell recognition of donor class I MHC peptides has been shown to actually play a role in the effector mecha- nisms of rejection [7].

Class I MHC antigens are more abundant than class II MHC antigens in the commonly transplanted organs [8]. Moreover, in heart, liver and pancreas of the rat, the only MHC class II-positive cells are transient, bone marrow-derived interstitial dendritic cells [9]. Similarly, the only MHC class II-positive structures in skin are macrophages and Langerhans cells [10]. Only kidney has MHC class II-positive parenchymal cells at the time of grafting [11]. Although MHC class II antigens are induced in the course of rejection, with varying degrees of facility in different organs and tissues [12-15], we were interested to test if priming to indirect T-cell recognition of a MHC class II peptide might also result in accelerated rejection responses.

3. Materials and Methods

3.1. Exper imenta l animals

Inbred BN (RTI"), WAG (RTlU), DA (RTlaVl), PVG (RT1 c) and LEW (RT1 l) male rats were obtained from Harlan-Olac, Bicester, Oxon.

3.2. Pept ide

The sequence of the peptide used in this study was taken from published data on the RT1.Db u class II molecule [16]. This corresponds to amino acids 60-79, which were calculated to be in the hypervariable a

SSDI0165-2478(94)00082-3 195

helix [17], and was chosen to maximise the chance of polymorphism with other strains. The sequence of the peptide used in the study was: Tyr-Arg-Asn-Lys-Gln- Lys-Glu-Phe-Met-Glu-Arg-Arg-Arg-Ala-Thr-Val-Asp- Thr-Tyr-Cys.

The peptide was purchased from Cambridge Re- search Biochemicals (Cambridge, UK) as a standard grade reagent. The peptide was dissolved in 0.1 M NHnHCO 3 and then desalted on PD-10 columns packed with G10 (Pharmacia, Uppsala, Sweden). The peptide containing fractions were pooled, freeze-dried and stored with desiccant at 4°C.

3.3. Peptide immunisation

All immunisations were with free peptide, unlinked to any carrier proteins. Rats received 50 /xg of peptide in Freund's complete adjuvant subcutaneously into each hind footpad at week 0, and 50 /zg of peptide in Freund's incomplete adjuvant at week 4.

3.4. Cell preparations

3.4.1. Lymph node cells Cervical, para-aortic, mesenteric and popliteal lymph

nodes were used. After washing, the cells were resus- pended at 2 X 10 6 cel ls /ml in RPMl-1640 supple- mented with 2 mM glutamine, 5 x 10 -5 M 2- mercaptoethanol, 100 U / m l penicillin, 100 ~ g / m l streptomycin and 5% heat-inactivated normal rat serum (Gibco, Paisley, Scotland), hereafter referred to as 'cul- ture medium'.

3.4.2. Depletion of adherent cells from lymph node cells Lymph node cells (LNC) at 108 cel ls /ml in Hepes-

buffered RPM1-1640 containing 1% heat-inactivated normal rat serum were applied to a sterile Sephadex G-10 (Pharmacia, Uppsala, Sweden) column previously equilibrated in this buffer at 37°C. After 30 min at 37°C the cells were eluted with one column volume of Hepes buffer.

3.4.3. Separation of CD4 + and CD8 + cell subsets Whole LNC were depleted of adherent cells as de-

scribed in the preceding section. All subsequent proce- dures were carried out at 4°C. For the CD8 ÷ prepara- tion, OX12 [18], OX35 [19] and W3/25 [20] antibodies were added to coat the B cells and CD4 ÷ cells. For the CD4 ÷ preparation OX12 and OX8 [21] antibodies were added to coat the B cells and CD8 ÷ cells. The anti- body-coated cells were removed using magnetic Dyn- abeads (Dynal, Wirral, Merseyside, UK). Purity of the preparations was assessed by flow cytometry and was 98-99% for CD4 ÷ T cells and 81-92% for CD8 ÷ T cells.

3.4.4. Preparation of antigen-presenting cells A single cell suspension was prepared at room tem-

perature from the spleens of normal rats. Contaminating red blood cells were removed with Tris-buffered NH4C1 and the cells exposed to 1500 rad of irradiation from a 137Cs source using a Gamma Cell 1000 apparatus (Iso- medix, NJ) at 100% dose for 1-2 min. The cells were then washed and resuspended at 2 X 106 cel ls /ml in culture medium.

3.5. Cellular proliferation assays

LNC (2 X 105) were incubated in round-bottomed 96-well tissue culture plates (Sterilin, Feltham, UK) in 200/zl volumes of culture medium. Incubations were at 37°C in 5% CO 2 in air. To some cultures, 105 irradi- ated APC were added. Cultures were stimulated with specific and irrelevant peptides at 10 /xg/ml with ConA at 10 / zg /ml as a positive control. Twenty-four hours before harvesting, 1 /zCi of [3H]thymidine in 20 /zl of culture medium was added to each well.

3.6. Indirect radioactive binding assays on microtitre plates

These were used to test for the presence of anti- peptide antibodies in sera obtained from peptide-im- munised animals. The assay involved peptide-coated wells in a 96-well polyvinyl chloride microtitre plate (Dynatech, Billingshurst, Sussex, UK) and 125I-labelled immunoadsorbant purified, rabbit F(ab')2 anti-rat F(ab')2 (RAR) [22] to detect binding of antibodies to the plate, as previously described in detail [7].

3.7. Skin grafts

Full-thickness grafts of abdominal skin, approxi- mately 1-2 cm 2 in area, were placed on the dorsolateral thoracic wall of recipients. The dressings were removed at day 6 and the grafts thereafter assessed daily. The day of rejection was taken as the day on which more than 90% full-thickness loss had occurred.

3.8. Kidney grafts

Recipients received a left orthotopic kidney graft using microvascular surgery techniques [23] followed by a right nephrectomy at day 7.

3.9. Cyclosporin A

Cyclosporin A powder was a kind gift of Sandoz (Basel, Switzerland). It was dissolved at 10 m g / m l in olive oil by heating to 60°C, stored at room temperature

196

TABLE 1

ANTIBODY RESPONSE a IN VARIOUS RAT STRAINS TO A

PEPTIDE b FROM THE RTl.DB” MOLECULE

Rat strain MHC haplotype Titre ’ at week

2 4 6

BN n 81 243 505

DA avl 4 13 117

PVG c 0 19 56

WAG u 0 0 4

LEW 1 0 0 0

a All rats received 50 pg of carrier-free peptide in Freund’s complete

adjuvant and a boost of 50 pg of peptide in Freund’s incomplete

adjuvant 4 weeks later. The 6-week bleed therefore represents the

secondary response.

b Amino acids 60-79.

’ Geometric mean titre of 3 rats. The titre is the reciprocal of the last

dilution to give 50% of maximal binding in solid-phase assays.

in the dark, and used within 2 weeks of preparation. It was given orally to the rats by gavage.

4. Results

4.1. Antibody studies

The provision of T lymphocyte help for the produc- tion of antibodies to carrier-free peptides requires recognition of the peptide by the surface Ig receptors on B lymphocytes, internalisation, and interaction of the peptide (or fragments of the peptide) with the class II molecule of the B lymphocyte. Thus, as previously discussed [4], antibody production to free peptide is a simple screening method for the potential of a peptide to interact effectively with the class II MHC molecules of any particular strain.

The peptide used in this study was chosen to corre- spond to the hypervariable region of the (Y helical domain of the /3 chain of the RTl-D” class II molecule. The results of immunising several rat strains with the peptide are shown in Table 1. The LEW and WAG strains produced very little or no antibody to the pep- tide. This could be a consequence of lack of polymor- phism between the WAG and LEW strains for this region of the RTl.Db gene or more likely a failure of interaction of the peptide with the class II molecules of the LEW strain. The DA and PVG strains produced weak primary and moderate secondary antibody re- sponses to the peptide. However, the BN strain gave strong primary and secondary antibody responses. Ex- amples of the titration of the antibody response in sera from BN and LEW rats are shown in Fig. 1.

The anti-peptide sera were checked for cross-reactiv- ity against the whole RTl.Db” molecule on WAG

Fig. 1. Antibody responses to the RTl-Db” peptide. BN (A) and

LEW (B) rats were immunised with the RTl.Db” peptide as de-

scribed in Materials and Methods. The titrations shown are for sera

from a representative rat taken 2 weeks after the boost and titrated on

plates with (0) and without (0) peptide. cpm refer to 1251-labelled

RAR.

spleen cells. However, no cross-reactivity was found (data not shown).

4.2. Cellular proliferation studies

Lymph node lymphocytes from immunised BN rats gave a strong proliferation response to the peptide, but immunised LEW rats and unimmunised BN rats did not respond. Fig. 2 shows that the response in the BN rat was found only in the CD4+ T-cell subset.

; 4-

,‘3-

02- / 1 r

0: ’ II /_

3 4 5 3 4 5

TIME (DAYS)

Fig. 2. T-cell proliferation studies. Lymph node lymphocytes taken

from BN rats immunised 10 days after the booster injection with the

peptide were separated into the CD4+ (A) and CD8+ (B) T-cell

subsets, and stimulated in vitro with the peptide at 10 pg/ml (0) or

medium alone (O), together with irradiated BN APC. [3H]thymidine

incorporation was measured at days 3, 4 and 5 as indicated.

197

TABLE 2 5. Discussion THE EFFECT OF PEPTIDE IMMUNISATION ON THE SUR-

VIVAL OF WAG ALLOGRAFTS IN BN RATS

Recipient Cyclosporin

immunisation A a

Graft b Survival

(days)

These studies demonstrate that a synthetic allogeneic MHC class II peptide able to stimulate CD4+ T cells is nevertheless not able to influence allograft rejection. The most likely explanation for this is that the synthetic peptide might not correspond to a naturally processed fragment of the RT1.D” p chain. The failure of WAG to BN allografts to stimulate T-cell responses to the peptide is consistent with this explanation. If this is the

case, the T-cell responses to the synthetic peptide are of no relevance to the indirect allorecognition response of BN rats to WAG RT1.D class II molecules.

Control ’

Peptide d

None

Control ’

Peptide d

No

No

No

Yes

Yes

Skin 9,9,10,10,10,11

Skin 9,9,10,10,10,11

Kidney 8,10,11,11

Kidney 17,21,22,23,27,28

Kidney 11,21,21,21,27,36

a Cyclosporin A was given orally at 5 mg/kg/day for 7 days from

the time of grafting.

b Grafts were performed lo-14 days after the booster injection.

’ Freund’s adjuvant only.

d RTl.Db” peptide, as in Materials and Methods.

4.3. Transplantation studies

BN rats were immunised with the RTl-Db” peptide and subsequently received skin or kidney allografts. It can be seen from Table 2 that the peptide immunisation did not influence skin allograft survival.

The model of kidney grafting involves right recipient

nephrectomy at day 7, so that the rat relies on the left kidney graft for survival from day 7. By day 7, how- ever, the graft is virtually completely destroyed in this and other strain combinations, so that this model is of no value for detecting sensitisation. We therefore gave rats a short course of cyclosporin A, to give the peptide immunisation an opportunity to demonstrate shortening of graft survival. In previous studies, cyclosporin A was shown to be less effective for prolonging allograft survival in pre-sensitised rats [24]. However, the peptide had no effect on kidney graft survival in the peptide-im- munised rats.

4.4. Immunisations with WAG skin and kidney allo-

grafts

Immunisations of BN rats with WAG tissues were performed to see if T cells responsive to the synthetic RTl.Db” peptide can be found following natural pro- cessing of the RT1.D” p chain. BN rats were given a WAG skin graft following several weeks later by a WAG kidney graft. Approximately 10 days after the kidney graft the cervical lymph nodes and spleen were removed and the cells were tested for proliferation to the RTl.Db” peptide. No proliferation was seen in any of 3 experiments, in contrast to the proliferation seen in this same model using DA to LEW grafts and RT1.Aavl class I peptides [6].

There are other possible explanations. Firstly, indi- rect T-cell recognition of class II molecules might not be as important as the response to class I molecules [7] for the rejection of skin allografts. However, indirect

recognition of RTl.Ba and RTl.Bb MHC class II polypeptide chains (presumably involving multiple T- cell epitopes in each case) has been shown to cause accelerated skin graft rejection [25]. Secondly, in the kidney graft experiments, any effect of the indirect recognition response against class II peptides might have been masked by cyclosporin A, as this drug has previously been shown to suppress very effectively indirect T-cell recognition responses [26].

Overall, these studies emphasise the importance of bearing in mind the possibility that indirect T-cell allo- recognition responses to synthetic MHC peptides might not be relevant to the rejection response to naturally processed MHC antigens.

Acknowledgements

This work was supported by the British Heart Foun- dation. G. Murphy was supported by the National Rid- ney Research Fund. G.J. Sawyer is a Colt Foundation Scholar.

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