CELLULAR
IMMUNOLOGY
Enhancement
19,
of Lymphocyte Dependent Antibody by Anti Allotype Serum C.
Service
318-327 (1975)
d’lnzmunothe’rapie
BONA,
Cytotoxicity
D. JUY, AND P. A. CAZENAVE
Expkrinzentale et d’lwn~~nochi~nic 75015 Paris. France Received
February
Awalytique,
Imtitut
Pasteur,
20,1975
Anti-allotype b4 and anti-allotype a3 antibody as well as heterologous anti-rabbit IgG enhanced the lymphocyte-dependent antibody cytotoxicity, in a system using chicken red blood cells (ChRBC) coated with rabbit anti-ChRBC antibody (a3/a3, b4/b5) as target cells and rabbit lymphocytes (a3/a3, b4/b4). No enhancement was observed with anti-allotype b6 antiserum, nor with heterologous anti-rabbit IgM, IgA, and Fc antibodies. Cytotoxicity mediated by spleen, bone marrow, and thymus lymphocytes was enhanced by anti-allotype antibody. The enhancement of cytotoxicity by anti-allotype antibody cannot be attributed to lymphocyte proliferation but is more likely related to the formation of an additional bridge between effector cell and target cell.
INTKODUCTION It is generally admitted that antibody-coated target cells are lysed by small lymphocytes which carry Fc receptors (1, 2). The anti-target antibodies seem to be restricted to the IgG class and require an intact Fc portion (3) which permits the establishment of a bridge between the killer cell (by means of its Fc receptor) and the target cell coated with IgG antibody (by means of its Fc fragment) ( 1) . In mice, it was demonstrated that the effector cell of lymphocyte-dependent antibody (LDA) cytotoxicity carriers neither T (theta) nor B (Ig) markers (4) but could be a member of a subset of bone marrow-derived lymphocytes bearing the Ca receptor (5). In other species, such as human, the passage of the lymphocytes on beads coated with anti-human IgG antibody could decrease the “K” cytotoxicity, as compared to the one of nonpurified lymphocytes (1). It is known that, in human, the lymphocytes which carry Fc receptors also carry Ig receptors (6). Keeping these observations in mind, we have studied the effect of an anti-allotypic antiserum on LDA cytotoxicity performed with rabbit lymphocytes and rabbit anti-ChRBC antibodies. The anti-allotypic antiserum used was directed against the allotypic pattern of both the Ig receptor of effector cells and of the anti-target IgG antibody. MATERIALS
AND METHODS
Four to six months old Uouscat rabbits (Garches) 318 Copyright C 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
were used in all experiments.
LYMPHOCYTE
Separation
ANTIBODY
and Purification
Spleen lymphocytes described (7).
DEPENDENT
CYTOTOXICITY
319
of Spleen Cells
were
separated
according
to
a technique
previously
Antisera
Rabbit (with phenotype a ( l-2-3+) b (4+5+6-) anti-chicken erythrocyte antibody was prepared according to Perlmann et al. (1). Anti a3 antiserum was prepared in rabbit with phenotype a( l-2+3-) anti b4 antiserum in rabbit with phenotype b(4-5+6+) and anti b6 antiserum in rabbit with phenotype b (4+5+6-) . The technique of immunization has been previously described (8). Goat anti-rabbit IgG, IgM, IgA were obtained from Hyland Labs. Goat anti-rabbit Fc serum was obtained by the immunization with Fc fragment prepared according to Porter (9) and crystallized 7~. This serum did not react with Fab fragment of rabbit IgG in radioimmunoassays. Cytotoxic
Assay
The cytotoxicity of lymphocytes was evaluated by the release of radioactivity from chicken red blood cells (ChRBC) labeled with Xr (10) (sodium chromate having a specific activity of 264 mCi/mg and prepared in Saclay). Spleen cells (2.5 X 106) were suspended in RPM1 1640 medium (Flow Lab.) supplemented with 10% fetal calf serum and were mixed during 18 hr at 37°C with labeled ChRBC (lo”), in the presence of either normal rabbit serum or rabbit anti-ChRBC antiserum. Unlabeled sheep red blood cells ( 107) were added to the mixture to prevent the spontaneous lysis of target cells (5). Specific enhancement of LDA cytotoxicity by anti-allotypic antiserum was tested by incubating the target cells previously sensitized with rabbit anti-ChRBC antibody (a3/a3, b4/b5) with effector cells (a3/a3, b4/b4) in either rabbit antiallotypic antisera or goat anti-rabbit Ig sera. All the sera used in the system were heat-inactivated. Nonspecific release of radioactive chromium was determined by incubating 51Crlabeled ChRBC in absence of lymphocytes or rabbit serum. Maximum release of radioactivity was measured under the same conditions, after addition of lO$ triton to the labeled erythrocytes. Cell lysis was expressed as the percentage of “ICr-specific release, calculated as follows : cpm Ab - cpm SK x 100, cpm T - cpm SR where cpm Ab represent the counts in the supernatants of the tubes containing rabbit serum (normal or anti-ChRBC, alone or in combination with either antiallotypic or anti-rabbit Ig antisera), cpm SR, the nonspecific release in control tubes, and cpm T. the radioactivity released after treatment with 10% triton. Blast
Transformation
of Lymphocytes
In these experiments, splenic lymphocytes (2.5 x 106) were incubated for 18 hr, with either 20% autologous serum or 20% rabbit anti-allotype b4 antiserum.
320
BONA,
JUY
AND
CAZENAVE
After 12 hr, 1 &i [3H]thymidine (1 Ci/mM, Saclay, France) was added. Radioactivity incorporated by the cells was evaluated, according to a previously described technique (11). RESULTS Optimal Conditions of Cytotoxic Assays Figure la shows an experiment in which 2.5 X lo6 rabbit spleen cells were incubated for 18 hr with lO%hRBC labeled with 51Cr and with various dilutions of an immune and normal rabbit serum. Optimal specific release of isotope was observed at serum dilutions of 1: 3200 and 1: 32,000. There was no detectable cytotoxicity at serum dilution of 1:320,000. Figure lb shows the effect of duration of incubation : significant cytotoxicity was observed after 2 hr, and increasing to a plateau that was reached at 18 hr. Results of an experiment in which cytotoxicity was measured as a function of the ratio of killer: target cells are presented in Fig. lc ; the optimal ratio was found to be 2.5 x 106: IO” (25 : 1). In all subsequent experiments on LDA, the following conditions were used: 2.5 X lo6 rabbit spleen cells for lo5 Yr labeled ChRBC, rabbit anti-ChRBC (or normal rabbit) serum diluted 1:32OO, incubation at 37°C for 18 hr. Effect of Anti-Allatypic
Antiserum
on LDA Cytotoxicity
As can be seen in Fig. 2, an enhancement of LDA was observed by the incubation of lymphocytes for 24 hr with various dilutions of anti-b4 antiserum. This enhancement was dose-dependent, being optimal at a l/5 dilution of anti-allotypic serum. In the presence of this optimal concentration of anti-ailotypic serum (l/S), the LDA cytotoxicity of spleen cells from several rabbits was significantly enhanced (Table 1). A weak %r release was observed in presence of normal rabbit serum, in some cases.
FIG. 1. Optimal conditions for LDA mediated by rabbit lymphocytes. Ordinate in all three parts is percentage of Tr released. (a) Effect of dilution of rabbit antiserum to ChRBC; (b) effect of duration of incubation; (c) effect of variation in ratio of effector cells : target cells.
LYMPHOCYTE
I
1
without anti b.q
ANTIBODY
I
112.5
1110
DEPENDENT
I
1150
321
CYTOTOXICITY
I
ll500
dilution c,f anti bd anti serum
allotypic
FIG. 2. Effect of dilution of anti-allotypic h4 antiserum on I,DA cytotoxicity rabbit (b4/b4) lymphocytes. (0) Control (without allotypic serum). ( 0-j preincubated with rabbit (phenotype b4/b5) anti-ChRBC antiserum ( l/32.000) (ChRBC preincubated with rabbit (phenotype b4/h5) normal serum (l/32.000). taneous release. (a) Total triton release.
Conzparison of [3H] Thymidine Incorporation and LDA Cytotoxicity Incubation of Rabbit Spleen Lymphocytes whh Anti-b4 Anfiserm
mediated hy “‘Cr-ChRBC - -) “Cr(
After 24 hr-
There are several reports that anti-allotypic antiserum induces blast transformation in rabbit lymphocytes. We have studied the relationship between [ 3H] thymidine incorporation and the level of LDA cytotoxicity after incubation of lymphocytes for 24 hr with anti-b4 antiserum or autologous serum (control). As can be seen in Table 2, no correlation was found between the enhancement of LDA cytotoxicity and duplication of lymphocyte DNA in the presence of antinllotypic serum. Sljecificity
of the Enhancement of LDA Cytotoxicity
Results from experiments in which anti-b6, or goat anti-rabbit IgG, IgM or IgA, or goat anti-Fc fragment of rabbit IgG antisera were used in the LDA test are presented in Table 3. As can be seen, anti-b4 and goat anti-rabbit IgG antisera enhanced whereas anti-b6, and goat anti-rabbit IgM and IgA inhibited LDA cytotoxicity.
322
BONA,
JUY
AND
CAZENAVE
TABLE
Number of rabbit studied
Control
assays
NRSa
1 b4 ANTISIGKJM
EFFECT OF ANTI-ALLOTYPE LYMPHOCYTE-DEPENDENT
ON KARRIT CYTOTOXICITY
ANTIBODY
Assays performed in presence of anti-b4 serum
ISb
NRS
IS
Spontaneous release
Total triton release
1
377 i lgc @)d
1108 f 103 (28.3)
379 f 6 (0)
2488 f 131 (81.8)
378 f
16
2957 f
183
2
600 f 12 (0)
8.56 f 61 (15.0)
878 i 26 (16.3)
139.5 f 4.5 (47.5)
607 f
36
2267 f
46
3
5.53 f 8 (0)
1117 f 51 (33.5)
959 f 16 (24.2)
2153 f 15.5 (94.6)
549 f
56
2245 f
109
4
316 f 8 (0.1)
1.521 f 61 (23.7)
346 f 11 (0.5)
3726 zk 32 (67.4)
323 f
15
5373 f
601
5
576 f 52 (4.0)
989 f 43 (21.9)
922 f 139 (19.0)
11.51 f 217 (28.9)
483 f
38
2794 f
213
6
366 f 21 (3)
855 f 9 (71)
270 f 24 (1.0)
1059 f 19 (87.9)
257 f
38
1191 f
61
0 NRS = normal rabbit serum (phenotype b4/b4). b IS = immune serum: rabbit anti-chicken red blood cells antibody Ecpm - average f SD of triplicate tubes. d ( ) = ‘% of specific Wr release.
TABLE
BETWEEN THE ENHANCEMENT OF LYMPHOCYTE-DEPENDENT AND [~HITHYMIDINE INCORPORATION OF SPLEEN CELLS 18 HR WITH ANTI-ALLOTYPE b4 ANTISERUM
Number of experiments
Control
WI
with autologous Wr
thymidine incorporation
serum
release
NR!+
ISb
a3/a3, b4/b5).
ANTIBODY INCUBATED
CYTOTOXICITY
2
COMPARISON
cultures
(phenotype
Cultures
with anti-allotypic
PHI
Wr
thymidine incorporation
serum release
NRS
IS
1
6244 f
1070”
377 f 18 (Old
1108 f 103 (28.3)
6958 f
268
379 f 6 (0)
2488 f 131 (81.8)
2
3643 f
470
600 f 12 (0)
856 i 61 (15)
3921 f
110
878 f 26 (16.3)
1395 f 45 (47.5)
a NRS = normal rabbit serum (phenotype b4/b4). b IS = immune serum : rabbit anti-chicken red blood cells antibody c cpm - average f SD of triplicate culture tubes. d ( ) = % of specific 61Cr release. For spontaneous and total triton number 1 and 2.
(phenotype
a3/a3, b4/b5).
release see Table 1, rabbits
LYMPHOCYTE
ANTIBODY
DEPENDENT
TABLE
323
CYTOTOXICITY
3
INFLUENCEOF ANTI-ALLOTYPIC AND ANTI-ISOTYPIC ANTIBODIES ON CYTOTOXICITYOF SPLEEN LYMPHOCYTES Cytotoxicity
assays performed
wm
in presence of Tr-ChRBC NRSa
Nil
preincubated
with IS*
316 f 8 (0)”
1521 xtz 61 (23.7) 3726 f 82 (67.4)
Anti-b4
antiserum
(l/S)
343 zk 41 (0.4)
Anti-b6
antiserum
(l/S)
346 zk 11 (0.5)
897 f 200 (11.4)
Goat anti-rabbit
IgG antiserum
(l/S)
566 f 49 (4.8)
2121 f 154 (35.6)
Goat anti-rabbit
IgiVI antiserum
(l/S)
1127 f 531 (15.9)
1530 f 265 (23.9)
Goat anti-rabbit
IgA antiserum
Goat anti-IgG
Q NRS b IS = ’ ( ) = and total
Fc fragment
= normal rabbit immune serum: ye specific Tr triton release =
Enhancement Lyn@hocytes
antiserum
754 f
339 i 35 (0.3)
(l/S)
1071 f 12 (21.8)
320 zk 18 (0)
(i/5)
37
(8.5)
serum (phenotype b4/b4). rabbit anti-chicken red blood cells antibody (phenotype b4/b5). release. In this experiment, spontaneous Wr release = 323 i 15 cpm 5373 f 601 cpm.
of Cytotoxicity Mediated with Anti-b4 and Anti-a3
by Bone Antisera
Marrow,
Thymw,
and Spleen
In these experiments, rabbit (a3/a3, b4/b4) bone marrow, thymus, and spleen cells were used as effector. In the presence of anti-b4 or anti-a3 antiserum, LDA cytotoxicity was significantly enhanced for all effector cells. Enhancement was least for thymus cells, in the case of anti-b4 antiserum. With anti-a3 antiserum, enhancement was more marked for bone marrow cells than for thymus or spleen cells (Table 4). It should be noted that no enhancement by anti-b4 antiserum could be observed in three experiments in which effector cells derived from rabbits of genotype b(4-5-6’) were used (Table 5). DISCUSSION Our results clearly show that incubation of rabbit lymphocytes with antiallotypic antibody can enhance LDA cytotoxicity. The allotypic patterns of a series and b series (13) are located, respectively, on the Fd fragment and on the light chain of rabbit immunoglobulins (reviewed by Porter, 14).
324
BONA,JUY
AND CAZENAVE TABLE
4
INFLUENCE OF ANTI-&AND ANTI-b4 ANTISERA ON CYTOTOXICITY OF RABBIT (a3/a3, b4/b4) BONE MARROW, THYMIC AND SPLEEN CELLS Origin of effector cells
B one marrow
Cytotoxic assays performed in presence of Wr-ChRBC NRSO Normal rabbit serum (control) (phenotype a3/a3, b4/b4) Anti-b4 antiserum Anti-a3
Thymus
antiserum
Normal rabbit serum (control) (phenotype a3/a3, b4/b4) Anti-b4 antiserum Anti-a3
Spleen
cpm
antiserum
Normal rabbit serum (control) (phenotype a3/a3, b4/b4) Anti-b4 antiserum Anti-a3
antiserum
preincubated with IS*
249 f 18 (0)” 325 f 18 (7) 352 f 22 (10)
525 f 44 (38) 654 f 32 (57) 638 f 20 (55)
281 f
321 f
33
294 f (3) 274 f 0) 262 f (0) 293 f
7
03)
(2) 10 25
502 zt 5 (35) 437 f 2
11
460 f
(25) 28
(28) 23
(3) 233 f 11 (0)
835 f 47 (84) 516 f 29 (37)
n NRS = normal rabbit serum (phenotype b4/b4). b IS = immune serum : rabbit anti-chicken red blood cells antibody (phenotype a3/a3, b4/b5). C ( ) = ye of specific Wr release. In this experiment the spontaneous Wr release = 267 f 16 cpm and total triton release = 947 f 13 cpm.
Three alternative hypotheses could therefore be entertained to explain this enhancement : (1) The enhancement reflects the proliferation of lymphocytes, subsequent to blast transformation by anti-allotypic antiserum. In our experiments, however, the cells were incubated for only 18 hr, and no significant increase in [3H]thymidine incorporation was observed for lymphocytes cultured with anti-b4 antiserum. (2) The enhancement is due to increased availability of Fc fragments for effector cells, following the interaction of anti-allotypic antiserum with the allotypic antiserum with the allotypic patterns of anti-target cells antibody. This hypothesis can be ruled out, since no enhancement would be observed for effector cells of b6/b6 phenotype, cultured in the presence of anti-b4 antiserum and ChRBC coated with b4/b5 antibody. (3) The enhancement is related to the formation of an additional bridge between killer and target cells through a crosslinking between the allotypic patterns of rabbit IgG anti-target antibody and the IgG receptor carried by killer cells (see Fig. 3). We favor this last hypothesis, as it can also explain the enhancement of LDA cytotoxicity by anti-isotypic anti-IgG antibodies. Our results are in agreement with the findings reported by Resch et al. (15) of an enhancement of LDA cytotoxicity by goat anti-rabbit IgG antibody or by the
LYMPHOCYTE
ANTIBODY
DEPENDENT
TABLE
325
CYTOTOSICITY
5
ABSENCE OF ENHANCEMENT OF ANTI-b4 ANTISERUM ON CYTOTOXICITY MEDIATED BY RABBIT b6/b6 LYMPHOCYTES Number of experiments
Cytotoxic assays performed in presence of
Nil 1 Anti-b4
antiserum
Nil
Kr-ChRBC NRSa 345 f 51 (0.2)C 376 f 48 (2.3)~ !I 351 f 3 ]
(0.6)
2 Anti-b4
antiserum
Nil 3 Anti-b4
n NRS * IS = c( ) = and total
cpm
antiserum
324 f 21 (0) 340 zk 25 (0) 335 f 13 (0)
preincubated
with 19
1171 f
61
(57) 965 f (47)
12
1253 f (62.7) 1011 f (46) 844 f (34.5) 766 f (29.2)
107 15 48 25
= normal rabbit serum (phenotype b4/b4). immune serum : rabbit anti-chicken red blood cells antibody (phenotype b4/b5). 70 of specific Wr release. In this experiment the spontaneous release = 342 f 26 cpm triton release = 1795 f 158 cpm.
ChRBC
FIG. 3. Mechanism of enhancement of LDA cytotoxicity by anti-IgG and anti-allotypic sera. (a) Normal condition ; (b) in p resence of goat anti-rabbit IgG; (c) in presence of anti-b4 antiserum; (d) in presence of anti-a3 antiserum.
326
BONA,
JUY
AND
C.4ZENAVE
l;(ab’)z fragment of tllis antiljotly Ijut liot with the I~& frqment of :~i~ti-rajjljit I$; antibody. Using a similar system in guinea pigs (guinea pig effector cells and guinea pig anti-target cells antibodies), Chapuis et al. (16) have reported an inhibition of LDA cytotoxicity in presence of anti-isotypic anti-guinea pig Ig antibodies. This discrepancy can be explained by the absence of IgG receptor on the surface of K cells in guinea pig, as has been demonstrated in mice (4). The fact that anti-Fc antibody did not influence the level of LDA raises the problem of immunochemical availability of Fc fragment of the IgG receptor on rabbit cells. In human, the IgG is buried in the membrane only by pFc portion of Fc fragment (17) while in rabbit it could be possible that only the Fab portion of IgG macromolecule is exposed. The fact that the anti-IgM and anti-IgA antibodies inhibited LDA cytotoxicity can be explained by the blockage of the Fc receptor of effector cells by the goat antibodies. Enhancement of LDA cytotoxicity by anti-allotypic antibody is an immunologically specific phenomenon : an enhancement was observed only with antiallotypic antisera directed against the IgG of both the anti-target antibodies and the receptor of effector cells. It should be pointed out that the cytotoxicity of both B and T-derived lymphocytes is enhanced by anti-allotypic antibody. This is in agreement with reports that rabbit thymocytes can mediate LDA cytotoxicity (18) and therefore carry Fc receptors but the enhancement induced by anti-allotype serum suggests also the presence of IgG on the surface of these lymphocytes. The existence of Ig on rabbit thymocytes is a controversial matter (19-25). Nevertheless, our data suggest that, in rabbits, the cell which mediates LDA cytotoxicity carries both IgG and Fc receptor and can be found in central lymphoid organs, such as thymus and bone marrow, as well as in peripheral lymphoid organs, such as the spleen. ACKNOWLEDGMENT The
authors wish to acknowledgethe excellent technical assistanceof Miss Edith Bourgeois. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Perlmann, P., Perlmann, H., and Wigzell, H., Transplant. Rev. 13, 91, 1972. Mac Lennan, I. C. M., Transplant. Kev. 13, 67, 1972. Mac Lennan, I. C. M., Clis E.zrp. Immune. 10, 275, 1972. Greenberg, A. H., Shen, L., and Roitt, I. M., Cliln. Exp. Zmmwaol. 15, 258, 1973. van Boxel, J. A., Paul, W. E., Franck, M. M., and Green, I., J. Zmmzlnol. 110, 1027, 1973. Dickler, H. B., and Kunkel, H. G., J. Exj. Med. 136, 191, 1972. Bona, C., Trebiciavsky, J., Anteunis, A., Heuclin, C., and Robineaux, R., Eur. J. Zmmunol. 2, 434, 1972. Brezin, C., and Cazenave, P. A., Zmrnzrnochemisfry, in press. Porter, R. R., Biochem. J. 73, 119, 1959. Chedid, L., Juy, D., and Bona, C., Immunol. Comm. 3, 477, 1974. Bona, C., Chedid, L., Damais, C., Ciorbaru, R., Shek, P. N., Dubiski, S., and Cinader, B., J. Zmmunol. 114, 348, 1975. Sell, S., and Gell, P. G. H., J. Exp. Med. 122, 423, 1965. Oudin, J., C. R. Acad. Sci. (Paris) 250, 770, 1960. Porter, R. R., Ann. Zmnzunol. (Inst. Pasteur) 125C, 85, 1974.
LYMPHOCYTE
ANTIBODY
DEPENDENT
CYTOTOXICITY
327
15. Resch, K., Gelfand, E. W., and Prester, M., J. 1www101. 112, 792, 1974. 16. Chapuis, B., and Brumner, K. T., 1st. Arc/t. Allergy 40, 321, 1971. 17. Fr$land, S. S., and Natvig, J. B., Stand. J. Zmmm~l. 1, 1, 1972. 18. Gelfand, E. W., Resch, K., and Prester, M., Eur. J. 1mm~~1o1. 2, 419, 1972. 19. Trebiciavsky, I., Bona, C., Anteunis, A., and Robineaux, R., Fnliu Viol. (Praha) 18, 30, 1972. 20. Coombs, R. R. A., Gurner, B. W., Janeway, C. A., Wilson, A. B., Gell, P. G. H., and Kelus, PI. S., Inwwnology 18, 417, 1970. 21. Pernis, B., Forni, L., and Amante, L., J. Exp. Med. 132, 1001, 1970. 22. Jones, G., Marcuson, E. C., and Roitt, I. M., h’ature (Loudon) 227, 1051, 1970. 23. Sell, S., and Sheppard, H. W., Sricnce 1’82, 586, 1974. 24. Chou, C. T., Dubisky, S., and Cinader, B., J. Exp. Med. 126, 305, 1967. 25. Stecher, U. J., and Thorbecke, J. G., Iuw~rrloloy~ 12, 975, 1967.
IMMUNOLOGY
Enhancement
19,
of Lymphocyte Dependent Antibody by Anti Allotype Serum C.
Service
318-327 (1975)
d’lnzmunothe’rapie
BONA,
Cytotoxicity
D. JUY, AND P. A. CAZENAVE
Expkrinzentale et d’lwn~~nochi~nic 75015 Paris. France Received
February
Awalytique,
Imtitut
Pasteur,
20,1975
Anti-allotype b4 and anti-allotype a3 antibody as well as heterologous anti-rabbit IgG enhanced the lymphocyte-dependent antibody cytotoxicity, in a system using chicken red blood cells (ChRBC) coated with rabbit anti-ChRBC antibody (a3/a3, b4/b5) as target cells and rabbit lymphocytes (a3/a3, b4/b4). No enhancement was observed with anti-allotype b6 antiserum, nor with heterologous anti-rabbit IgM, IgA, and Fc antibodies. Cytotoxicity mediated by spleen, bone marrow, and thymus lymphocytes was enhanced by anti-allotype antibody. The enhancement of cytotoxicity by anti-allotype antibody cannot be attributed to lymphocyte proliferation but is more likely related to the formation of an additional bridge between effector cell and target cell.
INTKODUCTION It is generally admitted that antibody-coated target cells are lysed by small lymphocytes which carry Fc receptors (1, 2). The anti-target antibodies seem to be restricted to the IgG class and require an intact Fc portion (3) which permits the establishment of a bridge between the killer cell (by means of its Fc receptor) and the target cell coated with IgG antibody (by means of its Fc fragment) ( 1) . In mice, it was demonstrated that the effector cell of lymphocyte-dependent antibody (LDA) cytotoxicity carriers neither T (theta) nor B (Ig) markers (4) but could be a member of a subset of bone marrow-derived lymphocytes bearing the Ca receptor (5). In other species, such as human, the passage of the lymphocytes on beads coated with anti-human IgG antibody could decrease the “K” cytotoxicity, as compared to the one of nonpurified lymphocytes (1). It is known that, in human, the lymphocytes which carry Fc receptors also carry Ig receptors (6). Keeping these observations in mind, we have studied the effect of an anti-allotypic antiserum on LDA cytotoxicity performed with rabbit lymphocytes and rabbit anti-ChRBC antibodies. The anti-allotypic antiserum used was directed against the allotypic pattern of both the Ig receptor of effector cells and of the anti-target IgG antibody. MATERIALS
AND METHODS
Four to six months old Uouscat rabbits (Garches) 318 Copyright C 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
were used in all experiments.
LYMPHOCYTE
Separation
ANTIBODY
and Purification
Spleen lymphocytes described (7).
DEPENDENT
CYTOTOXICITY
319
of Spleen Cells
were
separated
according
to
a technique
previously
Antisera
Rabbit (with phenotype a ( l-2-3+) b (4+5+6-) anti-chicken erythrocyte antibody was prepared according to Perlmann et al. (1). Anti a3 antiserum was prepared in rabbit with phenotype a( l-2+3-) anti b4 antiserum in rabbit with phenotype b(4-5+6+) and anti b6 antiserum in rabbit with phenotype b (4+5+6-) . The technique of immunization has been previously described (8). Goat anti-rabbit IgG, IgM, IgA were obtained from Hyland Labs. Goat anti-rabbit Fc serum was obtained by the immunization with Fc fragment prepared according to Porter (9) and crystallized 7~. This serum did not react with Fab fragment of rabbit IgG in radioimmunoassays. Cytotoxic
Assay
The cytotoxicity of lymphocytes was evaluated by the release of radioactivity from chicken red blood cells (ChRBC) labeled with Xr (10) (sodium chromate having a specific activity of 264 mCi/mg and prepared in Saclay). Spleen cells (2.5 X 106) were suspended in RPM1 1640 medium (Flow Lab.) supplemented with 10% fetal calf serum and were mixed during 18 hr at 37°C with labeled ChRBC (lo”), in the presence of either normal rabbit serum or rabbit anti-ChRBC antiserum. Unlabeled sheep red blood cells ( 107) were added to the mixture to prevent the spontaneous lysis of target cells (5). Specific enhancement of LDA cytotoxicity by anti-allotypic antiserum was tested by incubating the target cells previously sensitized with rabbit anti-ChRBC antibody (a3/a3, b4/b5) with effector cells (a3/a3, b4/b4) in either rabbit antiallotypic antisera or goat anti-rabbit Ig sera. All the sera used in the system were heat-inactivated. Nonspecific release of radioactive chromium was determined by incubating 51Crlabeled ChRBC in absence of lymphocytes or rabbit serum. Maximum release of radioactivity was measured under the same conditions, after addition of lO$ triton to the labeled erythrocytes. Cell lysis was expressed as the percentage of “ICr-specific release, calculated as follows : cpm Ab - cpm SK x 100, cpm T - cpm SR where cpm Ab represent the counts in the supernatants of the tubes containing rabbit serum (normal or anti-ChRBC, alone or in combination with either antiallotypic or anti-rabbit Ig antisera), cpm SR, the nonspecific release in control tubes, and cpm T. the radioactivity released after treatment with 10% triton. Blast
Transformation
of Lymphocytes
In these experiments, splenic lymphocytes (2.5 x 106) were incubated for 18 hr, with either 20% autologous serum or 20% rabbit anti-allotype b4 antiserum.
320
BONA,
JUY
AND
CAZENAVE
After 12 hr, 1 &i [3H]thymidine (1 Ci/mM, Saclay, France) was added. Radioactivity incorporated by the cells was evaluated, according to a previously described technique (11). RESULTS Optimal Conditions of Cytotoxic Assays Figure la shows an experiment in which 2.5 X lo6 rabbit spleen cells were incubated for 18 hr with lO%hRBC labeled with 51Cr and with various dilutions of an immune and normal rabbit serum. Optimal specific release of isotope was observed at serum dilutions of 1: 3200 and 1: 32,000. There was no detectable cytotoxicity at serum dilution of 1:320,000. Figure lb shows the effect of duration of incubation : significant cytotoxicity was observed after 2 hr, and increasing to a plateau that was reached at 18 hr. Results of an experiment in which cytotoxicity was measured as a function of the ratio of killer: target cells are presented in Fig. lc ; the optimal ratio was found to be 2.5 x 106: IO” (25 : 1). In all subsequent experiments on LDA, the following conditions were used: 2.5 X lo6 rabbit spleen cells for lo5 Yr labeled ChRBC, rabbit anti-ChRBC (or normal rabbit) serum diluted 1:32OO, incubation at 37°C for 18 hr. Effect of Anti-Allatypic
Antiserum
on LDA Cytotoxicity
As can be seen in Fig. 2, an enhancement of LDA was observed by the incubation of lymphocytes for 24 hr with various dilutions of anti-b4 antiserum. This enhancement was dose-dependent, being optimal at a l/5 dilution of anti-allotypic serum. In the presence of this optimal concentration of anti-ailotypic serum (l/S), the LDA cytotoxicity of spleen cells from several rabbits was significantly enhanced (Table 1). A weak %r release was observed in presence of normal rabbit serum, in some cases.
FIG. 1. Optimal conditions for LDA mediated by rabbit lymphocytes. Ordinate in all three parts is percentage of Tr released. (a) Effect of dilution of rabbit antiserum to ChRBC; (b) effect of duration of incubation; (c) effect of variation in ratio of effector cells : target cells.
LYMPHOCYTE
I
1
without anti b.q
ANTIBODY
I
112.5
1110
DEPENDENT
I
1150
321
CYTOTOXICITY
I
ll500
dilution c,f anti bd anti serum
allotypic
FIG. 2. Effect of dilution of anti-allotypic h4 antiserum on I,DA cytotoxicity rabbit (b4/b4) lymphocytes. (0) Control (without allotypic serum). ( 0-j preincubated with rabbit (phenotype b4/b5) anti-ChRBC antiserum ( l/32.000) (ChRBC preincubated with rabbit (phenotype b4/h5) normal serum (l/32.000). taneous release. (a) Total triton release.
Conzparison of [3H] Thymidine Incorporation and LDA Cytotoxicity Incubation of Rabbit Spleen Lymphocytes whh Anti-b4 Anfiserm
mediated hy “‘Cr-ChRBC - -) “Cr(
After 24 hr-
There are several reports that anti-allotypic antiserum induces blast transformation in rabbit lymphocytes. We have studied the relationship between [ 3H] thymidine incorporation and the level of LDA cytotoxicity after incubation of lymphocytes for 24 hr with anti-b4 antiserum or autologous serum (control). As can be seen in Table 2, no correlation was found between the enhancement of LDA cytotoxicity and duplication of lymphocyte DNA in the presence of antinllotypic serum. Sljecificity
of the Enhancement of LDA Cytotoxicity
Results from experiments in which anti-b6, or goat anti-rabbit IgG, IgM or IgA, or goat anti-Fc fragment of rabbit IgG antisera were used in the LDA test are presented in Table 3. As can be seen, anti-b4 and goat anti-rabbit IgG antisera enhanced whereas anti-b6, and goat anti-rabbit IgM and IgA inhibited LDA cytotoxicity.
322
BONA,
JUY
AND
CAZENAVE
TABLE
Number of rabbit studied
Control
assays
NRSa
1 b4 ANTISIGKJM
EFFECT OF ANTI-ALLOTYPE LYMPHOCYTE-DEPENDENT
ON KARRIT CYTOTOXICITY
ANTIBODY
Assays performed in presence of anti-b4 serum
ISb
NRS
IS
Spontaneous release
Total triton release
1
377 i lgc @)d
1108 f 103 (28.3)
379 f 6 (0)
2488 f 131 (81.8)
378 f
16
2957 f
183
2
600 f 12 (0)
8.56 f 61 (15.0)
878 i 26 (16.3)
139.5 f 4.5 (47.5)
607 f
36
2267 f
46
3
5.53 f 8 (0)
1117 f 51 (33.5)
959 f 16 (24.2)
2153 f 15.5 (94.6)
549 f
56
2245 f
109
4
316 f 8 (0.1)
1.521 f 61 (23.7)
346 f 11 (0.5)
3726 zk 32 (67.4)
323 f
15
5373 f
601
5
576 f 52 (4.0)
989 f 43 (21.9)
922 f 139 (19.0)
11.51 f 217 (28.9)
483 f
38
2794 f
213
6
366 f 21 (3)
855 f 9 (71)
270 f 24 (1.0)
1059 f 19 (87.9)
257 f
38
1191 f
61
0 NRS = normal rabbit serum (phenotype b4/b4). b IS = immune serum: rabbit anti-chicken red blood cells antibody Ecpm - average f SD of triplicate tubes. d ( ) = ‘% of specific Wr release.
TABLE
BETWEEN THE ENHANCEMENT OF LYMPHOCYTE-DEPENDENT AND [~HITHYMIDINE INCORPORATION OF SPLEEN CELLS 18 HR WITH ANTI-ALLOTYPE b4 ANTISERUM
Number of experiments
Control
WI
with autologous Wr
thymidine incorporation
serum
release
NR!+
ISb
a3/a3, b4/b5).
ANTIBODY INCUBATED
CYTOTOXICITY
2
COMPARISON
cultures
(phenotype
Cultures
with anti-allotypic
PHI
Wr
thymidine incorporation
serum release
NRS
IS
1
6244 f
1070”
377 f 18 (Old
1108 f 103 (28.3)
6958 f
268
379 f 6 (0)
2488 f 131 (81.8)
2
3643 f
470
600 f 12 (0)
856 i 61 (15)
3921 f
110
878 f 26 (16.3)
1395 f 45 (47.5)
a NRS = normal rabbit serum (phenotype b4/b4). b IS = immune serum : rabbit anti-chicken red blood cells antibody c cpm - average f SD of triplicate culture tubes. d ( ) = % of specific 61Cr release. For spontaneous and total triton number 1 and 2.
(phenotype
a3/a3, b4/b5).
release see Table 1, rabbits
LYMPHOCYTE
ANTIBODY
DEPENDENT
TABLE
323
CYTOTOXICITY
3
INFLUENCEOF ANTI-ALLOTYPIC AND ANTI-ISOTYPIC ANTIBODIES ON CYTOTOXICITYOF SPLEEN LYMPHOCYTES Cytotoxicity
assays performed
wm
in presence of Tr-ChRBC NRSa
Nil
preincubated
with IS*
316 f 8 (0)”
1521 xtz 61 (23.7) 3726 f 82 (67.4)
Anti-b4
antiserum
(l/S)
343 zk 41 (0.4)
Anti-b6
antiserum
(l/S)
346 zk 11 (0.5)
897 f 200 (11.4)
Goat anti-rabbit
IgG antiserum
(l/S)
566 f 49 (4.8)
2121 f 154 (35.6)
Goat anti-rabbit
IgiVI antiserum
(l/S)
1127 f 531 (15.9)
1530 f 265 (23.9)
Goat anti-rabbit
IgA antiserum
Goat anti-IgG
Q NRS b IS = ’ ( ) = and total
Fc fragment
= normal rabbit immune serum: ye specific Tr triton release =
Enhancement Lyn@hocytes
antiserum
754 f
339 i 35 (0.3)
(l/S)
1071 f 12 (21.8)
320 zk 18 (0)
(i/5)
37
(8.5)
serum (phenotype b4/b4). rabbit anti-chicken red blood cells antibody (phenotype b4/b5). release. In this experiment, spontaneous Wr release = 323 i 15 cpm 5373 f 601 cpm.
of Cytotoxicity Mediated with Anti-b4 and Anti-a3
by Bone Antisera
Marrow,
Thymw,
and Spleen
In these experiments, rabbit (a3/a3, b4/b4) bone marrow, thymus, and spleen cells were used as effector. In the presence of anti-b4 or anti-a3 antiserum, LDA cytotoxicity was significantly enhanced for all effector cells. Enhancement was least for thymus cells, in the case of anti-b4 antiserum. With anti-a3 antiserum, enhancement was more marked for bone marrow cells than for thymus or spleen cells (Table 4). It should be noted that no enhancement by anti-b4 antiserum could be observed in three experiments in which effector cells derived from rabbits of genotype b(4-5-6’) were used (Table 5). DISCUSSION Our results clearly show that incubation of rabbit lymphocytes with antiallotypic antibody can enhance LDA cytotoxicity. The allotypic patterns of a series and b series (13) are located, respectively, on the Fd fragment and on the light chain of rabbit immunoglobulins (reviewed by Porter, 14).
324
BONA,JUY
AND CAZENAVE TABLE
4
INFLUENCE OF ANTI-&AND ANTI-b4 ANTISERA ON CYTOTOXICITY OF RABBIT (a3/a3, b4/b4) BONE MARROW, THYMIC AND SPLEEN CELLS Origin of effector cells
B one marrow
Cytotoxic assays performed in presence of Wr-ChRBC NRSO Normal rabbit serum (control) (phenotype a3/a3, b4/b4) Anti-b4 antiserum Anti-a3
Thymus
antiserum
Normal rabbit serum (control) (phenotype a3/a3, b4/b4) Anti-b4 antiserum Anti-a3
Spleen
cpm
antiserum
Normal rabbit serum (control) (phenotype a3/a3, b4/b4) Anti-b4 antiserum Anti-a3
antiserum
preincubated with IS*
249 f 18 (0)” 325 f 18 (7) 352 f 22 (10)
525 f 44 (38) 654 f 32 (57) 638 f 20 (55)
281 f
321 f
33
294 f (3) 274 f 0) 262 f (0) 293 f
7
03)
(2) 10 25
502 zt 5 (35) 437 f 2
11
460 f
(25) 28
(28) 23
(3) 233 f 11 (0)
835 f 47 (84) 516 f 29 (37)
n NRS = normal rabbit serum (phenotype b4/b4). b IS = immune serum : rabbit anti-chicken red blood cells antibody (phenotype a3/a3, b4/b5). C ( ) = ye of specific Wr release. In this experiment the spontaneous Wr release = 267 f 16 cpm and total triton release = 947 f 13 cpm.
Three alternative hypotheses could therefore be entertained to explain this enhancement : (1) The enhancement reflects the proliferation of lymphocytes, subsequent to blast transformation by anti-allotypic antiserum. In our experiments, however, the cells were incubated for only 18 hr, and no significant increase in [3H]thymidine incorporation was observed for lymphocytes cultured with anti-b4 antiserum. (2) The enhancement is due to increased availability of Fc fragments for effector cells, following the interaction of anti-allotypic antiserum with the allotypic antiserum with the allotypic patterns of anti-target cells antibody. This hypothesis can be ruled out, since no enhancement would be observed for effector cells of b6/b6 phenotype, cultured in the presence of anti-b4 antiserum and ChRBC coated with b4/b5 antibody. (3) The enhancement is related to the formation of an additional bridge between killer and target cells through a crosslinking between the allotypic patterns of rabbit IgG anti-target antibody and the IgG receptor carried by killer cells (see Fig. 3). We favor this last hypothesis, as it can also explain the enhancement of LDA cytotoxicity by anti-isotypic anti-IgG antibodies. Our results are in agreement with the findings reported by Resch et al. (15) of an enhancement of LDA cytotoxicity by goat anti-rabbit IgG antibody or by the
LYMPHOCYTE
ANTIBODY
DEPENDENT
TABLE
325
CYTOTOSICITY
5
ABSENCE OF ENHANCEMENT OF ANTI-b4 ANTISERUM ON CYTOTOXICITY MEDIATED BY RABBIT b6/b6 LYMPHOCYTES Number of experiments
Cytotoxic assays performed in presence of
Nil 1 Anti-b4
antiserum
Nil
Kr-ChRBC NRSa 345 f 51 (0.2)C 376 f 48 (2.3)~ !I 351 f 3 ]
(0.6)
2 Anti-b4
antiserum
Nil 3 Anti-b4
n NRS * IS = c( ) = and total
cpm
antiserum
324 f 21 (0) 340 zk 25 (0) 335 f 13 (0)
preincubated
with 19
1171 f
61
(57) 965 f (47)
12
1253 f (62.7) 1011 f (46) 844 f (34.5) 766 f (29.2)
107 15 48 25
= normal rabbit serum (phenotype b4/b4). immune serum : rabbit anti-chicken red blood cells antibody (phenotype b4/b5). 70 of specific Wr release. In this experiment the spontaneous release = 342 f 26 cpm triton release = 1795 f 158 cpm.
ChRBC
FIG. 3. Mechanism of enhancement of LDA cytotoxicity by anti-IgG and anti-allotypic sera. (a) Normal condition ; (b) in p resence of goat anti-rabbit IgG; (c) in presence of anti-b4 antiserum; (d) in presence of anti-a3 antiserum.
326
BONA,
JUY
AND
C.4ZENAVE
l;(ab’)z fragment of tllis antiljotly Ijut liot with the I~& frqment of :~i~ti-rajjljit I$; antibody. Using a similar system in guinea pigs (guinea pig effector cells and guinea pig anti-target cells antibodies), Chapuis et al. (16) have reported an inhibition of LDA cytotoxicity in presence of anti-isotypic anti-guinea pig Ig antibodies. This discrepancy can be explained by the absence of IgG receptor on the surface of K cells in guinea pig, as has been demonstrated in mice (4). The fact that anti-Fc antibody did not influence the level of LDA raises the problem of immunochemical availability of Fc fragment of the IgG receptor on rabbit cells. In human, the IgG is buried in the membrane only by pFc portion of Fc fragment (17) while in rabbit it could be possible that only the Fab portion of IgG macromolecule is exposed. The fact that the anti-IgM and anti-IgA antibodies inhibited LDA cytotoxicity can be explained by the blockage of the Fc receptor of effector cells by the goat antibodies. Enhancement of LDA cytotoxicity by anti-allotypic antibody is an immunologically specific phenomenon : an enhancement was observed only with antiallotypic antisera directed against the IgG of both the anti-target antibodies and the receptor of effector cells. It should be pointed out that the cytotoxicity of both B and T-derived lymphocytes is enhanced by anti-allotypic antibody. This is in agreement with reports that rabbit thymocytes can mediate LDA cytotoxicity (18) and therefore carry Fc receptors but the enhancement induced by anti-allotype serum suggests also the presence of IgG on the surface of these lymphocytes. The existence of Ig on rabbit thymocytes is a controversial matter (19-25). Nevertheless, our data suggest that, in rabbits, the cell which mediates LDA cytotoxicity carries both IgG and Fc receptor and can be found in central lymphoid organs, such as thymus and bone marrow, as well as in peripheral lymphoid organs, such as the spleen. ACKNOWLEDGMENT The
authors wish to acknowledgethe excellent technical assistanceof Miss Edith Bourgeois. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Perlmann, P., Perlmann, H., and Wigzell, H., Transplant. Rev. 13, 91, 1972. Mac Lennan, I. C. M., Transplant. Kev. 13, 67, 1972. Mac Lennan, I. C. M., Clis E.zrp. Immune. 10, 275, 1972. Greenberg, A. H., Shen, L., and Roitt, I. M., Cliln. Exp. Zmmwaol. 15, 258, 1973. van Boxel, J. A., Paul, W. E., Franck, M. M., and Green, I., J. Zmmzlnol. 110, 1027, 1973. Dickler, H. B., and Kunkel, H. G., J. Exj. Med. 136, 191, 1972. Bona, C., Trebiciavsky, J., Anteunis, A., Heuclin, C., and Robineaux, R., Eur. J. Zmmunol. 2, 434, 1972. Brezin, C., and Cazenave, P. A., Zmrnzrnochemisfry, in press. Porter, R. R., Biochem. J. 73, 119, 1959. Chedid, L., Juy, D., and Bona, C., Immunol. Comm. 3, 477, 1974. Bona, C., Chedid, L., Damais, C., Ciorbaru, R., Shek, P. N., Dubiski, S., and Cinader, B., J. Zmmunol. 114, 348, 1975. Sell, S., and Gell, P. G. H., J. Exp. Med. 122, 423, 1965. Oudin, J., C. R. Acad. Sci. (Paris) 250, 770, 1960. Porter, R. R., Ann. Zmnzunol. (Inst. Pasteur) 125C, 85, 1974.
LYMPHOCYTE
ANTIBODY
DEPENDENT
CYTOTOXICITY
327
15. Resch, K., Gelfand, E. W., and Prester, M., J. 1www101. 112, 792, 1974. 16. Chapuis, B., and Brumner, K. T., 1st. Arc/t. Allergy 40, 321, 1971. 17. Fr$land, S. S., and Natvig, J. B., Stand. J. Zmmm~l. 1, 1, 1972. 18. Gelfand, E. W., Resch, K., and Prester, M., Eur. J. 1mm~~1o1. 2, 419, 1972. 19. Trebiciavsky, I., Bona, C., Anteunis, A., and Robineaux, R., Fnliu Viol. (Praha) 18, 30, 1972. 20. Coombs, R. R. A., Gurner, B. W., Janeway, C. A., Wilson, A. B., Gell, P. G. H., and Kelus, PI. S., Inwwnology 18, 417, 1970. 21. Pernis, B., Forni, L., and Amante, L., J. Exp. Med. 132, 1001, 1970. 22. Jones, G., Marcuson, E. C., and Roitt, I. M., h’ature (Loudon) 227, 1051, 1970. 23. Sell, S., and Sheppard, H. W., Sricnce 1’82, 586, 1974. 24. Chou, C. T., Dubisky, S., and Cinader, B., J. Exp. Med. 126, 305, 1967. 25. Stecher, U. J., and Thorbecke, J. G., Iuw~rrloloy~ 12, 975, 1967.
