Tissue Antigens (1977), 10, 323-329 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s)

HLA Antibodies Cytotoxic only for €3 Lymphocytes

P. Richiardi, P. Diotallevi, T. Mate$, A. 0. Carbonara and E. S. Curtoni Istituto di Genetica Medica dell’Universitd di Torino, Turin, Italy Of 28 unabsorbed sera from polytransfused individuals or multiparous women, which were not cytotoxic for total peripheral blood lymphocytes, 13 were found t o react positively against B cell-enriched suspensions. These 13 sera were further characterized after platelet absorptionelution, as well as by pretreatment with turkey anti-human-@,-microglobulin serum. Different patterns were found: five sera behaved as pure anti-B cell reagents; four seemed to contain different antibody populations, directed against both B cell determinants and HLA-A, -B or -C antigens; four only contained antibodies directed against HLA-A, -B or -C specificities. Absorption experiments with purified T and B lymphocytes, showed that these last sera, although noncytotoxic for T cells, can be absorbed by them. It was concluded that not all sera reacting only with B lymphocytes recognize specificities absent from T cells, and that all sera should be exhaustively absorbed with platelets before being tested as anti-B cell-specific reagents. Received for publication 15th April, accepted 19 April 1977

Most laboratories concerned with HLA serology are currently involved in the study of antigens present only on B lymphocytes which, by analogy with the murine “Ia”, are thought not to be associated with 0 2 microglobulin. Many sera containing anti-B lymphocyte antibodies also contain antibodies specific for other antigens (namely HLA-A, -B and -C, which are commonly called “SD” antigens); the SD antigens are present on all (or most) leukocytes and on platelets; they are associated, on the cell surface, with 0 2 -microglobulin. To discriminate between anti-B specific and antiSD specific antibodies, we have used three different approaches:

1)elimination of anti-SD antibodies by previously absorbing the sera with platelets (van Rood et al. 1975). A positive reaction still detected against B cells is considered to be due to anti-B cell specific antibodies. 2) pretreatment of B cell-enriched suspensions with a turkey anti-human-02 microglobulin serum (which is nor cytotoxic in the presence of rabbit complement (Bernoco et al. 1976)). Such cells can n o longer react with antibodies directed against PZ-microglobulin-associated structures (“bb” test: Bernoco et al. 1976); only antigens not associated with p 2 microglobulin will still react positively in cytotoxicity assays with the antibodies

This work was supported by C.N.R. “Centro di studio per l’lmmunogenetice ed Istocompatibilit 6‘’

3 24

RICHIARDI ET AL.

present in the serum. This technique, therefore, allows the use of unabsorbed antisera. 3) testing of B cell-enriched suspensions with sera which, even if unabsorbed, do not react in cytotoxicity with total peripheral blood lymphocytes (PBL) and are thus thought not to contain anti-SD antibodies. The aim of the present study was to investigate whether this last approach is correct in its assumptions and safe t o use. Materials and Methods Sera studied were obtained from individuals immunized by planned transfusions or from multiparous women. All of these sera were negative when tested in lymphocytotoxicity with the standard NIH technique (Brand et al. 1970), or with prolonged incubation. Panel: The sera were tested against a panel of PBL and B-enriched suspensions, from donors typed for HLA-A, -B, -C and sometimes -D factors.

The lymphocytotoxicity test used with both PBL and B-enriched suspensions was that agreed upon for the 7th International Histocompatibility Workshop: 1pl of the cell suspension (3,000 cells/pl) was incubated with 1p1 of serum for 1h at 20°C; 5 p1 of rabbit complement were then added and the total mixture incubated for 2 h at 2OoC. Dead cells were then stained with 5% Eosin Y and the reaction stopped with 3 7% formalin. B lymphocytes-enriched suspensions were

prepared by Ficoll sedimentation of sheep red blood cell (SRBC) rosettes, using the method of van Rood et al. (1975) with the following modifications: SRBC were pretreated with . 1 4 M AET; human PBL and SRBC were incubated for 15 min at 37"C,

then spun at 250g for 10min and incubated for a further 60min at O'C (Belvedere et al. 1977). The amount of Erosette-forming cells present in the enriched suspensions ranged from 5% to 15%. Purified T lymphocyte suspensions were collected from the sedimented E rosettes by hemolizing the SRBC with hypotonic treatment. EA-rosette-forming cells present in the purified suspensions represented about 5%. Absorptions and elutions from platelets were performed as described in previous papers (Richiardi et al. 1974, Belvedere e t al. 1975). One ml of serum was absorbed with 10" platelets carrying the relevant specificities. Absorptions with purified T and B lymphocyte suspensions were performed for 2 h at 2OoC. The amounts of absorbing cells ranged from 5 x lo6 to 100 x lo6 per ml of serum. The "bb" test was performed according to Bernoco et al. (1976). B cell-enriched suspensions (3,000 cells/pl) were incubated with a turkey anti-human-02 -microglobulin antiserum for 2 h at 2OoC, agitating every 15 min. The turkey antiserum had previously been controlled for lack of cytotoxic activity in the conditions of the test, in the presence of rabbit complement. After incubation, the treated cells were directly tested by the cytotoxicity test described above. Results Twenty-eight unadsorbed sera, negative against the specific immunizer in the standard lymphocytotoxicity technique, were

ANTIBODIES CYTOTOXIC FOR B LYMPHOCYTES

325

Table 1 ~

“bb” test

“7th Workshop test” Pattern

Serum:

a

Unabsorbed Platelet absorbed Eluate

b

Cells: Total PBL

Unabsorbed Platelet absorbed Eluate Unabsorbed Platelet absorbed Eluate

C

B-enriched

B-enriched

+ +

+ +

-

-

+ + + +

+ + -

-

+

Cytotoxicity test performed on sera before and after platelet absorption, and on their eluates. The cytotoxic test used has been carried out on cells both uncoated (“7th Workshop Standard test”) and previously coated with turkey anti-human-@,-microglobulin serum (“bb test”)

25

I

-

senm 21.10 absorbed by T lymphocytes serm 21.10 absorbed by B lymphocytes m-¤ eluate ol swum 21K) by T lymphocytes m--mekrate of SWM ZLlo absorbed by B lymphocytes 0-0

0--0

Figure 1. Doses (per &I of serum) of B and T lymphocytes necessary to absorb the cytotoxic activity against B cells. Absorptions have been performed both on whole serum and on its eluate from platelets. The cytotoxic activity is indicated by scores which take into consideration both the serum titer and the percentage of cells killed at each dilution.

!26

XICHIARDI El‘ AL.

dose of

absorbing cens t x l d )

Figure 2. Absorption experiments of the cytotoxic activity, against B cells, of serum 36.11 by T and B lymphocytes. The cytotoxic activity is indicated by scores which take into consideration both the serum titer and the percentage of cells killed at each dilution. The doses are per ~.rlof serum.

tested against a cell panel for lymphocytotoxicity. From each panel donor, both PBL suspensions and B lymphocyteenriched suspensions were separately tested and 13 of the sera always reacted negatively with PBL, but positively with the B-enriched suspensions of some donors. These 13 sera were further studied by absorption with platelets, elution from platelets and the “bb” test. Three patterns of results were observed (see Table 1) : a) Five sera (36.11, 30.29, 34.7, PR 10, PR 15) gave positive reactions against B lymphocytes (also after absorption with platelets) and no antibody reacting with B lymphocytes could be eluted from the platelets used for absorption. The sera were positive also on B cells previously coated with turkey anti-human$* microglobulin serum (“bb” test).

b) Four sera (51.29, 51.23, 35.20, PR 2) seemed to contain two antibody populations positive only against B cells: the first could not be absorbed with platelets and reacted also in the “bb” test; the second antibody population, on the other hand, could be absorbed by (and eluted from) platelets and inhibited in the “bb” test. These eluted antibodies, while positive with B-enriched lymphocytes, were not cytotoxic for total PBL. c) Four sera (21.10, 17.12, 3.48, 37.7), showed yet another pattern of reactivity. Every cytotoxic reactivity was removed by absorbing these sera with platelets. The platelet eluates were positive against B-cellenriched suspensions, but not against total PBL. When tested in the “bb” test, both the unabsorbed serum and the eluates gave negative reactions against B cells. These

327

ANTIBODIES CYTOTOXIC FOR B LYMPHOCYTES

Table 2 Pattern

Serum:

HLA antigen:

XZ

a a

36.11 30.29

Dw2 Dw5 Bw16

11.83 5.76

PR 2 51.29 51.23

Dwl Dw2 Bw16 Bw16

21.10 37.7

A10 A1 1

C C

P(Fisher test) .043

6.19 4.28 .0028 .00002

HLA antibodies cytotoxic only for B lymphocytes.

Tissue Antigens (1977), 10, 323-329 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission f...
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