Tisszle Antigens (1978), 11,443-448 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s)

False HLA-D Assignments May Be Caused by Cytotoxic Responder Lymphocytes Tom Kristensen and Fritz Jdrgensen Tissue Typing Laboratory, University Hospital, Aarhus, Denmark Lymphocytes from a healthy female repeatedly giving rise t o MLC-typing response against HLA-D homozygous typing cells of three different specificities were investigated for cytotoxic capacity by the direct CML technique. Testingagainst a panel indicated the presence of circulating cytotoxic lymphocytes with specificity towards HLA-A2. When tested against selected HLA-D homozygous typing cells, the pattern of CML reactivity closely resembled the pattern of MLCtyping responses, i.e. typing respmses were mostly restricted by the presence of HLA-A2 on the stimulator cells. This pattern was also found when time course studies of Cr-51 release were performed using experimental conditions identical t o ordinary MLC typing, but involving chromium-5 1 labeled, irradiated homozygous typing cells as targets. These studies indicate that the presence of in vivo educated cytotoxic lymphocytes among the responder lymphocytes may in some instances mimic typing responses. Such lymphocytes are thought t o lyse relevant stimulator lymphocytes prior to initiation of the proliferative response. Received for publication 29 September 1977, accepted 5 January 1978

Assignment of HLA-D phenotypes is classically made on the basis of Mixed Lymphocyte Culture (MLC) reactivity. For this purpose lymphocytes from the individual to be HLA-D typed are confronted with mitotically inactivated stimulator lymphocytes from highly selected individuals known to be HLA-D homozygous. N o or low reactivity in these cultures is thought t o imply that the responding lymphocytes carry an HLA-D determinant equal t o or resembling that of the homozygous typing cell (HTC). Thus, as distinct from most other typing systems, positive assignment is made on the basis of

a negative observation. Even if this inference is only reflecting the basic immunological concept of self-tolerance, it constitutes the major drawback of HLA-D typing by HTCs, since the interpretation of no or low MLC reactivity is ambiguous. Consistent with this, individuals have been identified who, by HTC typing, repeatedly exhibit more than two different HLA-D determinants. In The Sixth International Workshop on Histocompatibility, approx. 4% of the unrelated individuals investigated by HTC typing could be assigned more than two HLA-D determinants (Thorsby & Piazza 1975).Thomsen

This work was supported by the Danish Medical Research Council

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et al. (1976) have reported specific nonresponsiveness towards four distinct HLA-D determinants in an individual who could not have inherited and did not carry two of these determinants as judged by Primed Lymphocyte Typing (PLT). Apart from HLA-D compatibility between stimulator and responder, negative MLCs may tentatively be brought about by other factors: (i) genetic factors, such as homozygosity at an Immune-Response (IR) region leading to non-responsiveness towards certain determinants. In the mouse an IR region has been mapped centrally in the H-2 complex (Klein 1975), whereas IR regions have not yet been identified with certainty in humans, especially not in the HLA system. (ii) environmental factors, induced by e.g. microbial infection, pregnancy, bloodtransfusion, andlor allotransplantation: (a) specific tolerance t o certain determinants, (b) occurrence of specific suppressor cells or factors, (c) occurrence of specific cytotoxic cells. The object of this communication is t o draw attention to the last possibility, namely that the responder lymphocytes may be cytotoxic t o stimulator lymphocytes. As a consequence relevant stimulator lymphocytes are lysed before MLC proliferation is properly initiated, leading t o no or low responses and hence t o false assignmenu of HLA-D specificities. Material

This study concerns a healthy, 3 5-year-old female (HP) repeatedly and consistently yielding HLA-D typing responses against homozygous typing cells of three different specificities. There is no family history of

immunological disorders, nor of genetic diseases. HLA-A, -B, -C genotype: A l , B8, Cw-lA3, B7, Cw-. HP has a history of one pregnancy (1975) and no bloodtransfusions. After her pregnancy HP developed a weak lymphocytotoxic antiHLA-A2 antibody (titre 1/2 - 1/4). This antibody disappeared approx. 18 months after pregnancy and has not been present since then. Further, HP developed lymphocytotoxic lymphocytes with a specificity for HLA-A2 plus some as yet unidentified determinant(s). These cells were observed for the first time approx. 24 months after pregnancy and can still be demonstrated, although they have been absent in intervals of up to one month. Methods

HLA-D typing was performed using local HTCs and the technique of Jdrgensen & Lamm (1974). Typing was performed for the following specificities: Dwl ( 2 HTCs); Dw2 ( 2 HTCS); Dw3 (2 HTCs); Dw4 ( 2 HTCs); and Dw6 (2 HTCs). All HTCs were either included in the VIth International Workshop or have been closely matched t o workshop reference cells. Routine typing experiments involve approx. 20 responders and approx. 15 HTCs and include pool-stimulation by three different pools of three highly selected stimulators each. This set-up allows expression of the proliferative responses as stabilised, relative responses (SRR) (Ryder et al. 1975). A response below 40% SRR is considered as typing response. Direct CML testing was performed as described in detail elsewhere (Grunnet et al. 1975). In brief, lymphocytes from the individual t o be investigated are confronted

FALSE HLA-D ASSIGNMENTS

445

Table 1 HTC typing of HP HLA-D HLA-ABC Local

1 st test 2nd test 3rd test Mean

w2 wl 2,27 3 , ~ 3 5 3 , 7 2,7 330 170 136 216 12

3

0

0

15

9

Homozygous Typing Cells w3 w4 w6 1 , 8 1 , 8 2 , ~ 1 5 2 , ~ 1 5 2 , 9 , ~ 1 25 , ~ 4 0 , ~ 1 5 217 225 133 250 307 311

2

581 387 161

65 146 102

553 297 231

949 565 189

12 2 34

12 2 2

67 29 33

26 20 61

2

376

104

360

568

16

5

43

36

Responder cell = HP

for 6 h in duplicate cultures with Cr-51 labeled lymphocytes from a panel of selected individuals (ratio 50/1). The amount of lympholysis is expressed by a Cr-5 1 release percentage as calculated by the formula: (experimental - spontaneous) cpm x 100 (maximal - spontaneous) cpm After 6 h of incubation a release percentage above 10%is considered positive. The chromium-51 MLC assay was performed as a routine MLC assay using Cr-51 labeled, irradiated HTCs, i.e. 5 x lo4 responding lymphocytes versus 5 x lo4 Cr-51 labeled stimulating lymphocytes in a total volume of 150 /A. Experiments were performed as time-course studies and involved six independent, but identical, setups with supernatant harvesting after 2, 4, 6, 8 , 10 and 12 h of incubation. All combinations were performed in triplicates. Maximum release was defined by repeated (X 3) freeze thawing of the Cr-51 labeled stimulator cells alone, while spontaneous release was defined by the release in cultures involving Cr-5 1 labeled, irradiated stimulators versus autologous unlabeled and unblocked lymphocytes. Experimental time was limited t o 8-12 h by the spontaneous chromium-release.

Results Our results of repeated HLA-D typings of HP are given in Table 1. When responses below 40% SRR are considered as typing responses, HP should be assigned three different HLA-D phenotypes, i.e. HLA-Dwl, Dw4 and Dw6. B-lymphocyte typing using w7 antisera (VIIth Histocompatibility Workshop) indicated that HP possesses the HLA-DRwl determinant. Consequently, the responses against Dw4 and Dw6 typing cells may possibly be false. This finding and further typings of HP using a battery of Italian typing cells (a gift from Dr. G.B. Ferrara, Massa, Italy) revealed that the typing responses of HP were, in the majority of cases, restricted by the presence of the HLA-A2 antigen on the HTC. Normally, HP gives a rather low response t o the reference pools, hence the generally very high relative responses t o the Dw2 and Dw3 cells; furthermore, it is seen that t h e HLA-A2 positive Dw2 HTC stimulates less than the A2 negative Dw2 typing cell, this probably reflects the same phenomenon as operates in the Dw4 and Dw6 HTCs. Ordinary-direct C M L testing of HP against target lymphocytes from five selected HTC donors indicates the presence of circulating cytotoxic lymphocytes

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Table 2 Ordinary C M L chromium-51 release percentages of HP on five local HLA-D homozygous typing cells in two different experiments w4 w6 w2 w3 wl 2so 307 3 30 136 225 HLA-A, -B, -C A3;Bw35;Cw- A3;B7;Cw-A1;Bg;Cw- A2;BwlS;Cw- A2,9;Bw15;CWHLA-D

HTC No.

1st test 2nd test

3.7% 2.1%

0.3% 1.7%

2.3% 2.9%

Effector = HP indicates positive findings

a

Table 3 Time-course chromium51 MLC assay with HP on five chromium labeled HLA-D homozygous typing cells HTC No. Specificity: Incubation : 2h 4h 6h 8h 10h 12h

*

3 30

Dwl 0.0%

-0.5% 4.5% 5.1% 6.0% 4.4%

136 Dw2

225 Dw 3

250 Dw4

2.O% 1.3% 0.2% -1.3% 0.5% 1.5%

-0.1% -0.5% 3.9% 5.3%

-2.5% 8.4% 113.9361

* *

0.0%

4

*

307 Dw6 -2.5% 3.6% 3.1% 0.4%

120.1%1 118.3%

= release percentage above 10,considered positive = not done;spontaneous release 2 maximum release

directed against determinants present on some, but not all, HTCs (Table 2). HP lymphocytes significantly lyse HTCs 250 (Dw4) and 3 0 7 (Dw6); these findings fit in with the direct CML testing against highly selected panel-lymphocytes and may be seen to parallel the patterns of MLC typing responses. HP’s direct CML activity seems to be directed towards the HLA-A2 antigen. For the purpose of seeing whether these cytotoxic lymphocytes disclosed themselves in, and possibly interfered with, MLC typing as performed by HTCs, CML reactivity against the same HTCs was assayed in time-course experiments using Cr-51 labeled HTCs (Cr-51 MLC assay) (Table 3).

Not knowing the exact negative reference values, firm quantitative conclusions are not possible, but accepting a 10%level for positivity the qualitative conclusions closely resemble those reached by ordinary, direct CML testing (Table 3 cf. Table 2). Family testing revealed the father giving typing response t o the same HTCs as HP and in addition t o Dw3 HTCs. The Dwphenotyping results and the most likely HLA genotyping of the family appear in Table 4. These findings may be explained by assuming a paternal HLA-B, -D recombination in the uncle and further that HP is D w l homozygous. However, by DRw typing on B cells (Histocompatibility Testing 1977) DRwl is found in the mother (as in HP) but not in the father, which

I

447

FALSE HLA-D ASSIGNMENTS

Table 4 HLA typing of the HP family HTC typing: Dw

* *)

*)

P. Grandfather P. Grandmother P. Aunt P. Uncle Father Mother Sister HP

?

3,2? 3 2

1,3,4,6 1 3 1,4,6

HLA-Genotyping:*) A3, B7, DwlIA11, B5, DwX (deduced, n.i.) Aw19, B8, Dw3IA2, B12, Dw2? A l l , B5, DwX/Awl9, B8, Dw3 A3, B7, DwXIA2, B12, Dw2 A 3 , B7, D w l ( 4 , 6 ) I A w l 9 , B8, Dw3 A l , B8, DwlIA9, B18, DwZ Aw19, B8, Dw3IA9, B18, DwZ A3, B7, D w l ( 4 , 6 ) / A 1 , B8, D w l

HLA-C typing not performed

* *) P = paternal DwX and Z = unknown HLA-D alleles. n.i. = not investigated

might indicate that the Dwl assignment in the father is false. This will agree with MLC testing, as HP, functionally, is not D homozygous; she only elicits a weak response (i.e. probably typing response) in the mother, not in the father; neither do her cells function as typing cells when tested on a panel of Dwl-positive and -negative individuals. Furthermore, as the father stimulates the sister only weakly, but is stimulated by her, the MLC results in the family rather indicate an inclusion of the MLC determinant($ on the paternal A3, B7 haplotype in the determinant($ on the maternal A9, B18 haplotype. By ordinary direct CML testing on a selected panel the father’s cells disclosed cytotoxicity parallel to HP’s cells, but on a lower level.

Discussion The findings presented demonstrate a suggestive parallelism between “false” MLC-typing responses and CML reactivity against the HTCs involved. Whether cytotoxic lymphocytes present in the responder cell population are the direct cause of MLC negativity or whether their

presence reflects activation of other immune-regulatory factors, such as suppressor lymphocytes (cf. Thomsen et al. this issue) is not clear. On the basis of the results with time-course studies involving Cr-51 labeled HTCs, we tend, however, to favor the simple conclusion that in vivo primed cytotoxic lymphocytes present in the responder population may lyse relevant stimulator lymphocytes prior to the actual triggering of the responding clones. This mechanism is thus an alternative t o other explanations of MLC unresponsiveness, e.g. genetic non-responders and specific tolerance, but i t also opposes these, inasmuch as individuals exhibiting cytotoxic lymphocytes are not immunologically inert, but rather immunologically normal or even “high-responders”. Another theoretical cause of MLC-low responses would be the presence among the stimulator lymphocytes of cytotoxic cells active against relevant responder cells. This possibility can be ruled out in the case presented here, since all local HTCs have been screened for CML reactivity against selected panels and were, moreover, found t o be CML-negative against cells from HP.

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In the light of the number of reproducible and consistent MLC typings performed with these HTCs, the latter mechanism seems to be irrelevant for the present problem, although it may be relevant in relation t o other HTCs. We cannot at present offer any plausible explanation for the finding in the father, we are not aware of an adequate immune stimulation. Some time after the procurement of the batch of cells (frozen) for these experiments the father developed a classical case of erysipelas. It is tempting to speculate on some connection between the apparent anti HLA-A2 cytotoxicity and the occurrence of this disease, which is caused by type A streptococci, as it has been shown that streptococcal M protein cross-reacts with the HLA-A2 antigen (Hirata & Terasaki 1970). The typing pattern in these two related individuals may be fortuitous, but the findings may underline a possible genetic aspect in MLC responsiveness or unresponsiveness.

Histocompatibility Testing (1977).Ed. Bodmer, W.F. Munksgaard, Copenhagen. (In press). Jdrgensen, F. & Lamm, L.U. (1974) MLC-A micro-modification of the mixed leucocyte culture technique. Tissue Antigens 4, 482-492. Klein, J. (1975)Biology of the mouse histocompatibility-2-complex. Springer Verlag, New York. Ryder, L.P., Thomsen, M., Platz, P. & Svejgaard, A. (1975) Data reduction in LD-typing. Histocompatibility Testing 1975, pp. 557-562. Thomsen, M., Dickmeiss, E., Jakobsen, B.K., Platz, P., Ryder, L.P. & Svejgaard, A. (1978) Low Responsiveness in MLC Induced by Certain HLA-A Antigens on the Stimulator Cells. Tissue Antigens (this issue). Thomsen, M., Morling, N., Platz, P., Ryder, L.P., Staub-Nielsen, L. & Svejgaard, A. (1976) Specific lack of responsiveness to certain HLA-D (MLC) determinants with notes on primed lymphocyte typing (PLT). Transplant. Proc. 8,455-459. Thorsby, E. & Piazza, A. (1975) Joint report from The Sixth International Histocompatibility Workshop Conference. 11. Typing for HLA-D (LD-1 or MLC) determinants. Histocompatibility Testing 1975, pp. 414-458. Munksgaard, Copenhagen.

References Grunnet, N., Kristensen, T., Kornerup, H.J. & Kissmeyer-Neilsen, F. (1975) Direct cell mediated lympholysis in man. A test of allograft in human kidney recipients. Tissue Antigens 5 , 280-285. Hirata, A.A. & Terasaki, P.I. (1970) Crossreactions between streptococcal M proteins and human transplantation antigens. Science 168,1095-1096.

Address :

Tom Kristensen Tissue Typing Laboratory University Hospital DK-8000 Aarhus Denmark