studies on the role of hla-dr in macrophage-t cell interactions

Tissue Antigens (1981), 18, 217-231

Studies on the role of HLA-DR in macrophage-T cell interactions SUSAN BRIGHT and A. J. MUNRO

Immunology Division, Department of Pathology, University of Cambridge, Cambridge, England

The purpose of this paper is to illustrate the difficulties we have encountered in attempting to analyse the role of HLA-DR in the antigen-dependent co-operation between macrophages and T cells which leads to a T-cell proliferative response. We have adopted the two most commonly used approaches; attempted inhibition by anti-HLA-DR antisera and study of co-operation between cells of unrelated individuals, and have found both methods unsatisfactory. With the first method we found that anti-HLA antisera could inhibit proliferative responses in a non-specific manner. Both anti-HLA-A, B and anti-HLA-DR antisera could inhibit and this inhibition was largely Fc-dependent. Using pepsin-digested antisera we have no evidence for a unique role for HLA-DR in these proliferative responses. The second method, study of co-operation between cells of unrelated individuals, proved extremely difficult to analyse because of the background allogeneic reaction. Whether cells of two individuals appeared to co-operate to give an antigen-specific response depended on the number of cells used and the calculations applied to the data. However, it was clearly possible to demonstrate co-operation between DR different individuals. Received for publication 19 January 1981, revised, accepted 14 July 1981

In experimental animals it is well established that the Major Histocompatibility System (MHS) plays an important role in cell co- operation in the immune system (Munro & Waldmann 1978). In particular it is known that molecules coded for by the I region of the MHS - the Ia antigens - are involved in the presentation of antigen to T cells by macrophages which then leads to T cell activation, measurable by proliferation. This has been deduced from two experimental approaches. First, primed T cells will only co-operate with macrophages of an Ia type compatible with that of the priming environment (Rosenthal &

Shevach 1973, Thomas et al. 1977). This is MHS restriction. Secondly, antibodies directed against the Ia molecules have been shown to block such macrophage-T cell interactions (Shevach et al. 1972, Ruhl & Shevach 1975, Schwartz et al. 1976, Thomas et al. 1977).

In man, the HLA-DR locus codes for pro- ducts similar to the rodent Ia antigens in their biochemistry and tissue distribution (Bodmer 1978). It is expected that the HLA-DR antigens will have the same functional role as rodent Ia. Both of the experimental approaches outlined above, that is, studies on

000 1-28 15 i 8 1 /0902 17-15 $02.50/0 @ Munksgaard, Copenhagen

218 BRIGHT & MUNRO

MHS restriction (Bergholtz & Thorsby 1977, Hansen et al. 1978, Berle & Thorsby 1980, Rodey et al. 1979) and antibody inhibition (Bergholtz & Thorsby 1978, Geha et al. 1979), have been used by others to provide evidence for a primary role of HLA-DR in human macrophage-T cell co-operation. We have also tried these approaches and have found both techniques unsatisfactory. The object of this paper is to explain why our results have failed so far to demonstrate a unique role for HLA-DR in these experimental systems.

Material and methods

Cell cultures

Unseparated cells: Peripheral blood was taken from normal human volunteers, previously immunised to tetanus toxoid. The blood was defibrinated and mononuclear cells isolated in a standard fashion using dextran sedimentation and then centrifugation over FicoWmet- rizoate (Lymphoprep, Nyegaard). The basic proliferative assay was carried out with unseparated mononuclear cells. These were cultured in round-bottomed tissue culture plates (Linbro) at a density of lo5 lympho- cytesiwell, final volume 150 pl. The culture medium consisted of RPMI 1640 (Gibco) supplemented with 20% normal human serum (male AB), antibiotics (100 i.u./ml. penicillin, 100 pgiml streptomycin) and L-glutamine. Tetanus toxoid was added to some wells at the appropriate concentration and the cells incubated for 6 days at 37°C' in 5% CO, in air. There were 3 to 4 replicates for each treatment.

Macrophage-T cell interactions: In some experiments separate macrophage and T cell populations were used. These were prepared according to the methods of Bergholtz &

Thorsby (1977). Macrophage monolayers were prepared in the plates to be used for these culture experiments: lo5 mononuclear cells (about 10% monocytes) were added to wells of flat-bottomed tissue culture plates (Linbro) in culture medium. The cells were incubated for 1 h at 37"C, 5% C 0 2 and then the nonadherent cells removed and the wells washed once. The plates were then incubated for a further 18 h at 37"C, 5% COz and washed once more to remove nonadherent cells. At this stage most of the cells left adhering to the plate appeared to be macrophages and this preparation usually gave no proliferative response to antigen. The exact number of macrophages per well could not be standardised with this protocol. The approxi- mate number of macrophages was 104/well ranging from 5 x lo3 to 2 x lo4.

T cells were prepared in the following way: after dextran sedimentation of peripheral blood the leukocyte-rich supernatant was incubated with carbonyl iron and phagocytic cells removed using a magnet. The remaining cells were then incubated with IgG-coated ox red cells so that Fc-receptor bearing cells would form rosettes. This mixture was layered over FicoWmetrizoate and centrifuged in order to remove contaminating human red cells, ox red cells and rosettes. The interface layer was collected and usually contained less than 2% Fc rosetting cells and more than 80% sheep red cell rosetting cells. Generally this preparation (referred to as T cells) did not respond to antigen without the addition of macrophages. Occasionally there was a weak response presumably reflecting the presence of contaminating macrophages.

For culture, lo5 T cells were added to wells of flat bottomed tissue culture plates containing macrophage monolayers prepared as de- scribed above. The cells were cultured in a total volume of 150 p1 culture medium either with or without the addition of tetanus toxoid. As controls, the T cell or macrophage popula-

HLA-DR IN MACROPHAGE-T CELL INTERACTIONS 2 19

tions were cultured alone. The plates were incubated for 5 days at 37"C, 5% C02.

Measuring Proliferation: In all experiments, 6 h before the end of the culture, 20 p1 of RPMI 1640 containing 1 pCi of 3H- Thymidine (2Ci/mmol., Amersham. England) were added to the wells. At the end of the culture the cells were harvested onto glass-fibre filters and counted in a liquid scintillation counter.

Antigen

Tetanus toxoid was obtained from R. I. V. Bilthoven, The Netherlands, and from the Wellcome Research Laboratories, Beck- enham, Kent. This was diluted in culture medium and added to the wells at a final concentration of 0.5 or 1 i.u./ml.

H L A typing of donors

All donors were typed for alleles of the HLA-A, B and DR loci using a panel of in- ternationally defined typing antisera (His- tocompatibility Workshops 1977 and 1980) and standard tissue typing techniques. Some of this typing was kindly performed by Dr. V. Joysey and members of the tissue typing laboratory, Addenbrooke's Hospital Cam- bridge. The donors were not HLA-D typed by homozygous typing cells.

H L A antisera used for inhibition studies

The following pregnancy antisera were used to attempt to inhibit tetanus toxoid induced proliferation.

Antisera Specificity Origin

SE 40311.3 DR 1, 2, 6 Van Rood PL 44306.5 DR 4, A2, B15 CB 22199.3 DR 2, B8 ZO 12385.2 DR 7, A29 ME 49526.1 DR 5

f

KZ 26182.5 DR 1 MO 46104.1 DR 3, B8, BW6 "

MA A2 + Joysey BA DR4 + co polyspecific DR

In all cases the sera were heat inactivated, dialysed against phosphate buffered saline (PBS) and sterilised by high speed centrifugation before use. In most cases, as indicated in the text, dialysis was more extensive than this and mimicked the pepsin digestion protocol (see below) without the addition of pepsin.

Monoclonal anti-HLA antibodies

In some experiments a monoclonal anti-HLA reagent was used. This was: W6/32 (serum ascites) anti-HLA-A, B, C (Barnstable et al. 1978) (Sera Labs). Control: normal mouse serum.

Pepsin digestion of H L A antisera

The method of pepsin digestion is taken from Jonker & de Rooy-Doyer 1980. The HLA pregnancy antisera were dialysed overnight at 4°C against 0.1M sodium acetate buffer pH 4.0. Pepsin, dissolved in the same buffer, was then added at a final concentration of 2%. The mixture was incubated for 16 h at 37"C, centrifuged to remove visible precipitate and the supernatant dialysed for 3 h vs. 0.1M Tris buffer pH 8.0 and then overnight versus PBS at 4°C. Normal human serum was also pepsin digested as a control. Other aliquots of the antisera were treated in parallel but without the addition of pepsin as further controls.

The pepsin-digested antisera were no longer cytotoxic but were able to inhibit the cytotoxicity of intact antisera (Table 4). These inhibition experiments were carried out with B cells prepared by a standard sheep red cell rosetting technique. The results show that the F(ab)* fragments retain binding activity and,

220 BRIGHT & MUNRO

although it is impossible to measure absolute amounts of F(ab), antibody by this assay, that four fold excess of digested over intact antibody blocks completely the cytotoxic activity of the intact antibody. This would suggest that at least 25% of the binding activity has been retained after pepsin digestion.

Platelet absorption of HLA antisera

In some experiments the H L A antisera were platelet absorbed before being used in inhibition studies. Platelets were prepared from the

appropriate donor using a standard protocol (Bright e t al. 1978) although no azide was added to the storage medium. After storage for at least one month the platelets were washed 'once in PBS and E D T A and then once in PBS alone before being added to the H L A antisera. 4 x lo8 platelets were used to absorb 100 p1 of antiserum in 2 aliquots, each absorption for 20 minutes a t 4°C. The platelets were removed by spinning in a microfuge and the antisera stored at -20°C before use. Platelet absorbed normal human serum was used as a control.

Table 1 The proliferative response to teranus toxoid can be inhibited by any anti- HLA antiserum which reacts with the donor cells

D o n o r H L A Type A n t i s e r u m Shared R e s p o n s e to T e t a n u s S p e c i f i c i t y Toxoid

A 2 , 8 7 , B 2 1 (86) DR2, DR4

A 2 , 87, 8 8 , (86) DR1, DR3

A l , A 3 , B 7 , BE DR2. DR6

A l , A 2 8 , 814 , B 1 5 DR1, DR4

A 2 , B 7 , B 1 5 DR4

NHS CB a n t i DR2, 88 PL a n t i DR4, A 2 , B 1 5 20 a n t i DR7. A 2 9 MO a n t i DR3, BE, B 6

NHS 20 a n t i DR7. A 2 9 MO a n t i DR3, B 8 , B 6 CB a n t i DR2, BE

NHS SE a n t i DR1, 2 , 6 PL a n t i DR4, A 2 , 815

NHS KZ a n t i DR1 PL a n t i DR4, A 2 , B 1 5

NHS PL a n t i DR4, A 2 , B 1 5

- DR2 DR4, A2

B 6 -

- -

DR3, BE, B 6 BE

- DR2, 6

-

- DR1 DR4, B 1 5

- DR4, A 2 , 815

11551 (9.35 2 0.28) 3 2 2 * ( 5 . 7 5 + 0.35) 2 5 8 * ( 5.55 2 0.08)

10727 ( 9 . 2 8 2 0 . 3 2 ) 2 0 3 6 * ( 7 . 6 2 2 0.05)

4617 (8.44 0.23) 4835 (8.48 5 0.07)

360*(5.88 5 0.57) 8 2 6 * ( 6 . 7 2 2 0.54)

16019 (9.68 0.31) 5130*(8.54 5 0 . 2 7 )

14421 (9.58 2 0.31)

3381 (8.13 + 0 . 3 1 ) 1 7 6 * ( 5 . 1 7 5 0.83) 2 2 1 * ( 5 . 3 9 2 0.59)

6147 ( 8 . 7 2 5 0 . 2 2 ) 399*(5.99 2 0.86)

Results expressed as the arithmetic mean cpm 'H-Thymidine incorporation (geometric mean* 1 S.D.). NHS: normal human serum. Final concentration of test serum is 1/15. *: significant inhibition. P < 0 . 1 %.

HLA-DR IN MACROPHAGE-T CELL INTERACTIONS 2 2 1

Protocol for studies on antibody-induced inhibition of proliferation to antigen

In most experiments the HLA pregnancy antisera (or the monoclonals) were added directly to the wells of unseparated cells at the beginning of the culture with tetanus toxoid. The amount of antiserum added varied as indicated in the text but the final serum concentration remained at 20% when possible. In all cases control wells contained the same concentration of normal human serum (male AB) which had been treated in the same way as the HLA antiserum. That is, heat inactivated and then dialysed, pepsin digested or platelet absorbed. Normal mouse serum was the control for W6l32. There were also control wells with HLA antisera but without tetanus toxoid, in no case was proliferation above background seen in these wells. In some experiments, separated macrophage and T cell preparations were used and in others, antigen-pulsed cells. The significance of any inhibition seen was calculated using log, transformed data and analysis of variance. Results show the arithmetic mean and the log, transformed data.

Antigen-pulsing

In some experiments, antigen-pulsed cells were used. Unseparated mononuclear cells were incubated with tetanus toxoid (5 i d m l ) for 2 h in tissue culture tubes at a concentration of 2 x lo6 lymphocytes/ml in culture medium. The cells were then washed thoroughly 3 times and set up in round- bottomed tissue culture plates in the standard way. The proliferation seen must have been due to antigen taken up by the cells because the remaining antigen concentration, calculated by the dilution factor, is not enough to provoke cell proliferation. In some experiments HLA antisera or control sera were present during the pulsing.

Results

Inhibition of proliferative responses by H L A antisera

Addition of anti-HLA antisera to cultures of human mononuclear cells responding to tetanus toxoid often gave substantial inhibition. This inhibition was specific in the sense that only antisera directed against determinants present on the cells inhibited. However, inhibition was obtained with the "wrong" anti-HLA-DR antisera if they contained appropriate anti-HLA-A, B specificities (Table 1). To confirm that both anti-HLA-A, B and anti-HLA-DR antibodies could inhibit the tetanus toxoid induced proliferative response we showed that the inhibitory activity of some, but not all, antisera could be removed by platelet absorption (Table 2). This table

Table 2 The inhibifory activity of some, but not all, anti- H L A antisera for the tetanus toxoid proliferative response can be removed by absorbing with platelets.

Serum Response to Tetanus

29324 (10.29 2 0.27) NHS

MA 1012 ( 6.92 2 0.83)

NHS 63712 (11.06 2 0.19)

NHS (abs) 47602 (10.77 t 0.05)

MA (abs) 33889 (10.43 5 0.10)

BA (abs) 6138 ( 8.72 +0.08)

Results expressed as arithmetic mean cpm 3H-Thymidine incorporation (geometric mean * 1 S.D.). NHS: normal human serum. abs.: absorbed with platelets. Final concentration of test serum is 1 / 1 5 . BA (abs) gives significant inhibition compared to the NHS (abs) control, MA is no longer inhibitory after absorption. P < 0.1%.

15 Tissue Antigens 18. 4

222 BRIGHT & MUNRO

also illustrates one of the problems encountered in this study which was that non-specific platelet absorption affected the ability of a serum to support a proliferative response (see normal human serum control). Problems of this sort lead us to the conclusion that all test serum treatment must be accompanied by a control serum treated in exactly the same way. When the appropriate controls were used it was still apparent that the inhibitory effect by some pregnancy antisera on cell proliferation could be removed by platelet absorption thus indicating that anti-HLA-A, B antisera could inhibit such responses (Table 2). This was confirmed by the use of a monoclonal anti-HLA-A, B, C reagent W6/32 (Barnstable et al. 1978) which also substantially inhibited the proliferative response of cells to tetanus toxoid (Table 3).

We have frequently observed complete inhibition of the proliferative response to tetanus toxoid using anti-HLA-DR antisera specific for a single allele on heterozygous

Table 3 The proliferative response to tetanus toxoid can be inhibited by monoclonal anti-HLA-A, B, C: W6/32.

w6/32 conc. Response to Tetanus

- (normal mouse serum 1/150) 32749 (10.39 5 0.08)

1/150 3259 ( 8.09 5 0.24)

1/300 3370 ( 8.12 _+ 0.571

1/600 7445 ( 8.92 20.21)

1/1200 13732 ( 9.53 2 0 . 3 6 )

Results are expressed as the arithmetic mean cpm 3H-Thymidine incorporation (geometric mean k 1 S.D.). W6/32: Barnstable et al. 1978.

cells (Table 1). This, together with the observed inhibition by anti-HLA-A, B antisera, suggests that the mechanism of inhibition does not depend on the specificity of the

Table 4 Pepsin digested antisera will specifically inhibit cytoroxicity of untreated antisera

Donor Type: A 2 , All, B7, BE, DR2, DR3 B ce l l s

Antibody: CB anti-DR2, 88

Dilution digest: N 1/2 1/4 1/8 1/16 Pepsin NHS

dilution 2nd antibody: N 60 60 80 90 100 90

1/2 30 50 80 90 100 99

1/4 15 30 50 80 99 90

1/e 10 15 50 60 80 40

1/16 10 10 20 40 50 10

- 10 10 10 10 10 10

Results are expressed as the mean % dead cells: standard “B cell” microcytotoxicity test. Pepsin digestion according to the method of Jonker & de Rooy-Doyer (1980). NHS: normal human serum.

HLA-DR IN MACROPHAGE-T CELL INTERACTIONS 223

Table 5 The proliferative response to tetanus toxoid can not be readily inhibited by pepsin-digested anti- HLA-antisera

Donor Type

A1,28,B14,15 DR 1 , 4

A2 I B7 I 15 DR4

A2,B7,211B6 DR2,4

A2.B7,BEIB6 D R 1 I 3

P e p s i n - t r e a t e d An t i se rum

2/15 NHS

2/15 KZ anti-DR1

2/15 PL anti-DR4,A2,B15

4/15 NHS

4/15 KZ + PL

2/15 NHS

2/15 PL

1/15 NHS

1/15 CB anti-DR2,BB

1/15 PL

1/15 20 anti-DR7,A29

1/15 MO a n t i DR3,B8,6

4/15 NHS

4/15 CB

4/15 CB + PL

1/15 NHS

1/15 CB

1/15 PL

1/15 CB + PL

1/15 NHS

1/15 MO

1/15 KZ

1/15 KZ + MO

1/15 CB

Shared S p e c i f i c i t y

DR1

DR4 ,B15 -

DR1 + DR4,B15

DR2

DR4 ,A2 -

B6

- DR2

DR2 + DR4,A2

- DR2

DR4, A2

DR2 + DR4,A2

DR3, B8 ,B6

DR1

DR1 + DR3,B8,6

B 8

Response t o T e t a n u s

5258 (8.5750.33)

4948 (8.5120.23)

5665 (8.6420.24)

5656 (8.6420.80)

2442 (7.80+_0.34)

8271 (9.0220.27)

5922 (8.6920.19)

37849(10.54+0.12)

38712(10.56+0.12)

27810(10.2350.12)

31069(10.34+0.25)

3 59 3 4 ( 10.4 950.12 )

49712(10.8120.13)

20833* (9.9450.38)

30839(10.34+0.21)

18754 (9.8420.27)

5024 (8.5220.61)

5799 (8 .6720.53)

2489* (7.8220.58)

22828(10.04+0.26)

18832 (9.8420.55)

23031(10.05+0.36)

22980(10.04+0.52).

9362 (9.1450.56)

Results are expressed as the arithmetic mean cpm 'H-Thymidine incorporation (geometric mean ? 1 S.D.). + : X/15: proportion of the final well volume which consists of the added serum. *: significant inhibition p < 0.1%. NHS: normal human serum, also pepsin treated. This table can be directly compared with Table 1 because the sera for those experiments were treated in exactly the same way as these antisera, dialysed etc. without the addition of pepsin.

I S

224 BRIGHT & MUNRO

fnhihition of rhe proliferarive response of antigen-pulsed cells is Fc-dependent

antisera but rather on effector mechanisms recognising antibody of any specificity bound to the surface of the cells.

Consequently, the inhibition experiments were repeated using pepsin-digested antisera and pepsin-digested control sera. Pepsin-digested antisera were first checked for binding activity by showing that they could inhibit cytotoxicity of intact antibody (Table 4).

Table 5 shows that pepsin-digestion of both anti-HLA-A, B and anti-HLA-DR antisera removes the bulk of the inhibitory activity seen with the undigested antibodies. This was also true for combinations of pepsin-digested anti-HLA-DR antisera and when these reagents were used at a four-fold higher concentration than the undigested controls. In most

Table 6

experiments no inhibition at all was seen with pepsin-digested antisera although in some significant but incomplete and inconsistant inhibition was seen. This residual inhibition could be due to some intact antibody remaining in the preparations.

It is possible that pepsin-digested antisera are not inhibitory when antigen is continuously present in a culture because the activity of the antiserum degrades faster than the antigen. To check this we used antigen-pulsed cells. However, again we found that although the appropriate anti-HLA-DR antiserum would inhibit pulsed cells completely it was not inhibitory after pepsin-digestion whether the antiserum was present continuously or during the antigen pulsing (Table 6).

Donor: A2, B7, B15, DH4

Serum Treatment

NHS

PL anti-DR4, A2, B15

NHS

PL

2x N H S , pepsin

2x PL, pepsin

2x NHS, pepsin

2x PL, pepsin

Antigen

continuous

pulsed

continuous

pulsed

0 ,

Response to Tetanus

6147 (8.72 5 0.22)

399*(5.99 2 0.86)

8669 (9.07 2 0.31)

141*(4.95 2 0.38)

8271 (9.02 2 0.27)

5922 (8.69 2 0.19)

5759 (8.66 2 0.65)

3485 (8.16 2 0.61)

NHS: normal human serum. *: significant inhibition. p < 0.1%. Results are expressed as arithmetic mean cpm 'H-Thymidinc incorporation (geometric mean f 1 S.D.). Pepsin: added sera have first been pepsin treated by the standard method. The final concentration of test, untreated serum is 1/15 and of pepsin treated serum is 2/15.


Co-Gperation between the macrophages and T cells of two individuals in the response to tetanus toxoid

In summary, it would appear that the bulk of the inhibition by anti-HLA antisera of the proliferative response by mononuclear cells to tetanus toxoid is mediated via recognition of the Fc portion of antibody of any specificity bound to the surface of the cells. The mechanism of this Fc dependent inhibition is not known. Other results (not shown) indicate that the inhibition occurs only if the antisera are added in the first 4 days of the culture period and that there is no antigen specificity. Responses to antigens other than tetanus toxoid can be similarly non-specifically inhibited. Finally, we have been unable to demonstrate a specific repeatable inhibition by Fab'2 anti- HLA-DR reagents.

Macrophage- T cell interactions

An alternative way of studying the role of the MHS in human systems is to ask if T cells from primed individuals will co-operate with mac-

Table 7

rophages which are HLA incompatible to give an antigen dependent proliferative response. These experiments inevitably have to be done in situations where the T cells can respond to the allogeneic macrophages in the absence of added antigen - essentially an MLR. Thus, one is trying to see if a response of T cells to a given specific antigen can occur over the allogeneic reaction. Two approaches have been adopted by different authors to cope with this situation. The first is to reduce the allogeneic reaction. This can be achieved by limiting the number of T cells and/or macrophages and reducing the time of incubation (Bergholtz & Thorsby 1977). The second approach is to al- low a vigorous allogeneic reaction to occur and then to try to study the specific antigen response above this (Hansen et al. 1978). In both cases calculations are made to obtain an index of response enabling experiments performed on different days to be directly compared. These calculations involve a correction

Tetanus Toxoid Concentration (i.u./ml)

0 0 . 5

114 2 37 b SB~M~' 98 40

GK Ms 147 5 92 472 2 339

SB TC 131 t 36 187 f 58

GK T 156 2 15 300 2 lk3

SB Ms + SB T

SB Ms + GK T 2,883 5 635 31,413 2 902

250 2 163 4,746 2 2,998

GK MS + SB T 4,667 2 955 20,283 f 1,952

GK MS + GK T 128 2 80 40,129 2 7,334

a: DR types: SB 214, GK 2!6. b: results expressed as cpm 3H-Thymidine incorporation,

results are arithmetic means k 1 S.D. c: Ms: macrophages T: T cells.

1

116 f 39

916 2 778

130 5 41

317 2 80

4,826 2 1,558

22,526 f 8,216

25,484 2 7,960

46,042 5 3,590

226 BRIGHT & MUNRO

Table 8 Apparent co-operation betwecan macrophages and T cells of different individuals in the proliferative response to tetanus can depend on the number of co-operating cells

T Cell Donor Macrophage (MI Donor

JG l/-a JG l/-a

SB 2/4

RS 41-

GK 2/6

DF 1/3

RS 4/-

SB 2/4

GK 2 /6

EP 1/-

DF 1 / 3

DF 1 / 3

GK 2/6

Proliferative Response of T + M T + M + tetanus

842 53436*

221 ! 30056'

16728 45814*

6233 ! 6004

228 7799*

17025 17263

1095 ! 4696*

213 31784'

207 I ! 24965'

45010 48315

13214 ! : 44348*

3878 15068*

2986 I ! 18572'

276 5979*

234 I! 11392*

2980 12667*

6426 ! I 19731*

Results are expressed as the arithmetic mean cpm 'H-Thymidine incorporation. Significant increases upon the addition of tetanus are indicated by *: p < 1%. The mean response of the T cell populations alone to tetanus toxoid was 1106 cpm and of the macrophage populations alone was 306 cpm. a: DR types of T cell or macrophage donors. !: 5 x I04Tcells/well instead ofstandard lo5 !!: approx. 5 X 103macrophages/well instead ofstandard lo4.

for any allogeneic reaction to obtain the responses due to specific antigen. We have used both approaches and would like to make two general points. First, the apparent amount of antigen-specific response depends on the can occur.

method adopted and the calculation used. Second, whichever method or calculation is adopted, co-operation between macrophages and T cells of HLA-DR different individuals

HLA-DR IN MACROPHAGE-T CELL INTERAmIONS 227

Table 7 shows the full results from a macrophage-T cell co-operation experiment to illustrate the type of data obtained with our experimental protocol. In this case there is an obvious increment above the allogeneic background when the specific antigen is added indicating that the macrophages and T cells of these two donors can co-operate. Table 8 shows how the apparent tetanus-specific response can vary with the method used. For example, with the combination SB/JG if steps are taken to reduce the allogeneic reaction by decreasing the number of T cells there is no increment when tetanus is added. However, if the allogeneic reaction is allowed to occur by increasing the number of cells used, then a tetanus-specific response also appears. These two individuals are DR different. In contrast, in the experiment between RS and SB (half identical) and GK and EP (DR different) antigen-specific co-operation appears to occur with the lower cell doses but not with the higher. Finally, the combination GK/DF (DR

different) co-operate to give a tetanus-specific response with any dose of cells used.

In order to try and get an overall picture of the effect of DR compatibility on macrophage-T cell co-operation it is necessary to get some index of response so that experiments performed on different days can be directly compared. Most authors adopt a form of relative response (Bergholtz & Thorsby 1977, Hansen et al. 1978): comparing the response to specific antigen obtained between allogeneic T cells and macrophages to that of the syngeneic combination with the same T cell donor. The background “MLR” is sub- tracted before this comparison is made. Table 9 illustrates how this form of calculation can lead to bias. A high MLR means a higher variance so that the response with the addition of antigen may not be significantly different. However, subtraction of two large numbers can give a value which compares favourable with the lower syngeneic response. This point is illustrated with the combination RS/DF

Table 9 Measurement of a relative response is not an appropriate way of assessing co-operation between allogeneic macrophages and T cells

a

T C e l l Donor Macrophage (MI Donor P r o l i f e r a t i v e Response T + M T+M+ Tetanus

RS 4/-‘ RS 4/-‘ 204 5140*

DF 1 /3 10069 14755

AM 3/6 AM 3/6 1606 66503*

m 3/7 4546 33006*

b

of Relative Response

103%

79%

100%

42%

a: Results expressed as the arithmetic mean cpm 3H-Thymidine incorporation. b: Relative response calculated according to the method of Bergholtz & Thorsby (1977):

(T + M + tetanus) - (T + M): allogeneic (T + M + tetanus) - (T + M): syngeneic

c: DR types of T cell or macrophage donors. *: Significant increase in response upon the addition of tetanus. p < 0.1%. RS and DF show no significant response to tetanus but give a higher relative response than AMOH, which do show significant co-operation.

228 BRIGHT & MUNRO

(DR different) where a good relative response is obtained although there is no significant difference upon the addition of antigen.

The more usual stimulation index, obtained by dividing the response to antigen by the background, is not appropriate because the higher the MLR (the greater the D R difference) the lower the index can be because of the maximum limits of the tissue culture con- ditions. A bias is therefore introduced.

We concluded that there is no appropriate way of calculating data such as this where two proliferative responses are occuring in the same wells.

The experiments shown in this section illustrate how the apparent co-operation between macrophages and T cells of two individuals in the generation of an antigen-specific response varies according to the method adopted in the experiment and to the calculation applied to the results. It is possible to make a strong case for the role of HLA-DR with our data, hut equally, taking other data and other calculations into account, we can make a case for HLA-DR playing no role at all. The only clear statement we can make from this system is that there are D R different individuals who co-operate no matter which way the experiments are done and no matter which calculation is used. Table 8 shows an example of this.

Discussion

The purpose of this paper is to illustrate the difficulties we have encountered in attempting to analyse the role of HLA-DR in the antigen-dependent co-operation between macrophages and T cells. We have adopted the two most commonly used approaches; attempted inhibition by anti-DR antisera and study of co-operation between cells of unre-

lated individuals, and have found both methods unsatisfactory.

With the first method we found that the antigen-specific proliferative responses of T cells could be inhibited by anti-HLA-A, B as well as by anti-HLA-DR antisera. The inhibition was specific in as much as antisera would only inhibit if the appropriate determinant was expressed by the cell. However, it is unlikely that the specific reaction per se caused the inhibition but rather the reaction of antibody with cell surface antigens activated a non-specific inhibitor mechanism. This would explain our observations that complete inhibition could be obtained by anti-HLA-A, B or by anti-HLA-DR antisera directed against only one determinant with cells from a heterozygous individual and the fact that the mechanism appears to be Fc dependent.

It is difficult to know why the results of some other authors are not compatible with our findings. O n e group (Bergholtz & Thorsby, 1978) d o see inhibition by anti-HLA-A, B antisera as we do, however another group (Geha et al. 1979) d o not. Different antisera d o vary in their capacity to inhibit and lack of inhibition by one reagent cannot be used to prove that another is inhibiting specifically. Antisera will differ in strength, class and the sites where they attach to cells. This may explain why Engleman et al. (1980) could get n o or only marginal inhibition with saturation binding of monomorphic mouse anti-DR monoclonal antibodies whereas it is easy to get complete inhibition with much weaker pregnancy alloantisera. Human alloantibodies may activate the non-specific inhibitory effects more efficiently than heteroantisera. Non-specific, Fc dependent inhibition by anti-HLA antisera has also been observed in the mixed lymphocyte reaction (Jonker & de Rooy-Doyer 1980). The mechanism of the non-specific inhibition is unclear. It could involve steric hindrance,


antibody-dependent cell-mediated cytotoxicity or generation of suppressor cells. Broder et al. (1980) have found that an antiserum to HLA-DR will suppress pokeweed mitogen stimulation of human B cells and that this suppression is due to the generation of T suppressor cells. We did not try experiments like those of Bergholtz & Thorsby (1978) and Rodey et al. (1979) in which antisera were added to partially DR sharing mixtures of macrophages and T cells and were shown to give the best inhibition when directed against the shared DR determinant. These experiments suggests that in some circumstances specific inhibition can occur by blocking of the molecules involved in cell cooperation.

The inhibition of antigen-specific T cell proliferation using anti-Ia antisera was first observed in experimental animals. The results of these experiments were less ambiguous than the results we have obtained in man. There are probably two reasons for this. First, the inhibitory effects activated non-specifically by intact antibody may be less effective. Second, inhibitory antibodies are usually raised against the cells of the strain used for the experiments. In general such antisera contain antibodies to multiple la specificities, including public and private determinants. In man we are trying to block T cell proliferation using alloantisera made in response to cells from an unrelated donor. These antisera are selected to define single private DR specificities and consequently may be less effective in inhibiting antigen presentation. The same argument might apply to the observation that monoclonal antibodies to guinea pig Ia antigens are less effective at inhibiting T cell responses that alloantisera (Burger & Shevach 1980).

The conclusion from this first section of our work is that we have no evidence that the HLA-DR polymorphism plays a unique role in antigen presentation by macrophages to T cells. The biochemical and biological

similarities between HLA-DR and Ia molecules from rodents make it extremely likely that the DR molecules play a similar role in antigen presentation as Ia molecules. This is supported by the finding of HLA-DR restricted T cell lines in man as have been found in mouse (Kurnick et al. 1980, Sredni et al. 1980). However, we could not detect complete and consistent inhibition with F(ab), anti-DR antisera in contrast to the strong inhibition we saw with intact reagents. It is possible that the digested reagents are considera- bly weaker than the intact antibody although they were able to completely block the cytoxicity of intact antibody when used in 4 x excess (Table 4). Alternatively, the strong inhibition by intact antibody could be mediated by nonspecific mechanisms as outlined above and once these are eliminated then blocking by steric hindrance would not cover all the potential cooperating determinants. Other loci, homologous to DR, which are known to exist (Lampson & Levy 1980) may play an important role. In addition, it may be impossible to completely block the presentation of complex antigens using antisera to private DR specificities. The parts of the DR molecules involved in antigen presentation are not known and it is possible that presentation of some antigenic determinants involves parts of the DR molecules which do not carry the private specificities.

The second method of studying the role of HLA-DR in macrophage-T cell interactions in man is to ask if co-operation can be obtained between macrophages and T cells of DR different individuals. This protocol has been adopted by many authors to look at responses to a variety of antigens and the overall conclusion has been that these interactions are HLA-DR restricted (Bergholtz & Thorsby 1977, Hansen et al. 1978, Rodey et al. 1979, Berle & Thorsby 1980). We have not attempted a comprehensive series of experiments in which macrophages and T cells from

230 BRIGHT & MUNRO

a large number of different donors have been surveyed. However, our study has highlighted some problems with these experimental systems and we would like to make two points. First, the data obtained with these experimental protocols is difficult to analyse because of the background proliferation evoked by the allogeneic interaction (MLR). There is no satisfactory way to calculate the data to make comparisons between experiments. Second, it is common to observe co-operation between D R different macrophages and T cells by testing the interactions over a range of cell doses. In some of our experiments co-operation was best seen with lower cell doses - we interpret this to be due to domination of the culture by a vigorous MLK when higher cell numbers were used. In other experiments, co-operation between DR different donors could be best seen with higher cell concentra- tions. We interpret this as follows: with DR different donors there will be fewer T cell clones that can be activated, only those T cells that see antigen in the context of a shared (public or common, not private) D R epitope and thus a proliferative response will not be detected if the macrophage or T cell concentration is limiting. Hirschberg et al. (1980) using vascular endothelial cells as antigen- presenting cells also find that totally allogeneic cells can present antigen if cell numbers are increased sufficiently. Other authors (Bergholtz et al. 1980) have observed co- operation through cross reactive -presumably public determinants - and the known cross reactive groups explain most of the results of Table 8. We therefore agree with those authors that the private D R specificity is not the only restriction element. This is in contrast to other work (Hansen et a]. 1978, Rodey et al. 1979) where no or very little cross reaction was seen. A final problem with this system is that purified macrophage and T cell populations, however carefully prepared, will have some contaminating cells o f the other type

whose function may be enhanced by an ongoing allogeneic reaction.

We have outlined some of the problems involved in studying the role of HLA-DR in the induction of a proliferative response by human T cells to antigen. We feel that until further work has been reported on the effect of pepsin-digested monoclonal anti-DR antibodies (and mixtures of these) and on the production of a range of antigen-specific T cell clones, the role of the private, polymor- phic HLA-DR specificities in macrophage-T cell interactions must remain open.

Acknowledgments

We would like to thank Professor J. J. van Rood and Dr. V. Joysey for their generous gifts of antisera. We would like to thank the Medical Research Council for support and Nick Carter for technical assistance.

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Address: Dr. Susan Bright Department of Pathology University of Cambridge Tennis Court Road Cambridge CB2 1QP England

studies on the role of hla-dr in macrophage-t cell interactions

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