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Antibodies to T cell receptors and to histocompatibility antigens
CELLULAR
12,422-428 (1974)
IMMUNOLOGY
Antibodies
to T Cell Receptors and to Histocompatibility
Antigens1
H. RAMSEIER Division
of Experimental Microbiology, Microbiology, University Received
Departme& of Zurich
of Medical
November 14, 1973
The injection of 10’ parental strain spleen cells into adult Fl hybrid mice resulted in the formation of two serum activities: anti-receptor antibodies and alloantibodies. To demonstrate the respective roles of T and B cells in the elicitation of these serum activities, parental strain lymphoid cell suspensions containing varying proportions of T and B cells, or consisting only of T or B cells, were employed as inocula for. Fl mice. The results indicated that the injection of pure T cells led to the formation of antireceptor antibodies only, while the injection of pure B cells resulted exclusively in formation of alloantibodies, suggesting that anti-receptor antibodies were structures elicited by T cell receptors. Alloantibodies appear to be formed as a consequence of the interaction of B cell receptors with alloantigen.
INTRODUCTION The reaction between receptors of lymphoid cells and histocompatibility antigens is highly specific. It can be interfered with by two equally specific manipulations: blocking of receptors by anti-receptor antisera (1, 2) or blocking of transplantation antigens by alloantisera (2, 3). It is known that the injection of lymphoid cells from spleens or nodes of normal (unsensitized) animals of parental origin into appropriate adult Fl hybrid hosts results in the formation within the same serum of both anti-receptor antibodies and of alloantibodies (2). While it appears likely that alloantibodies are ultimately formed by bone marrow-derived (B) cells, it is not certain whether anti-receptor antibodies are formed in response to receptors present on thymus-derived (T) cells or to those present on B cells. Two observations make it likely that anti-receptor antibodies are directed against T cell receptors. First, thymus lymphocytes activated for alloantigens by in viva passage through irradiated recipients represent high numbers of antigen-sensitive T cells; they also elicit formation of anti-receptor antibodies (4). Second, in the test system employed to measure antigenic recognition, the PAR assay, only T cells, but not B cells, are essential (5). To establish the critical roles of T and B cells in the initiation of anti-receptor antibodies and of alloantibodies, a study was undertaken in which lymphoid cells 1 Abbreviations used in this paper : B cells, bone marrow-derived cells; BBS, balanced salt solution ; PAR, product of antigenic recognition; T cells, thymus-derived cells; TDL, thoracic duct small lymphocytes ; T.TNDL, thymus cells activated for alloantigens and harvested as small lymphocytes from thoracic duct. 422 CopyrightQ All
rights
1974 by of reproduction
AcademicPress, Inc. in any
form
reserved.
ALLOANTIGEK
of different origins raise Fl sera. The clusively against T as a consequence of
Am
T CELL RECEPTOR ANTIRODIES
423
and with varying contents of T and B cells were employed to results indicated that anti-receptor antisera were directed excell receptors, whereas alloantibodies appeared to be formed the interaction of B cell receptors with transplantation antigens. MATERIALS
AND
METHODS
Anbmls and lymphoid cells. Mice of inbred strains CBA, BALB/c and Fl hybrids of these strains with C57BL/6 were employed as donors of lymphoid cells. Nude mice (nu/nu)were kindly donated by Dr. B. Kindred; they were animals backcrossed to BALB/c mice for 7 generations. CBA thoracic duct small lymphocytes (TDL) and CBA thymus lymphocytes activated for C57BL/6 alloantigens and harvested from the thoracic duct (T.TDL) were gifts from Dr. J. Sprent. Other cells employed were from spleen, bone marrow and thymus. All cell suspensions were prepared in Hanks’ BSS (Gibco, New York, NY) without serum but fortified with 100 IU penicillin and SO pg/ml streptomycin. This medium was also used to maintain cells in short term cultures (PAR assay). Sensitization. To obtain cells actively sensitized to alloantigens, spleen cell suspensions were prepared from CBA mice 21 days after full-thickness C57BL/6 skin grafts. Treatment of spleen cells with anti-theta a,nttierunz and covtzplement. Anti-OC3H antiserum was prepared according to Cerottini et al. (6). Spleen cells at a concentration of 2 x lo7 were suspended in 1: 10 anti-BC3H and incubated for 30 min at 37°C under tissue culture conditions (5% CO2 in moist air). They were then centrifuged (130s for 8 min) and resuspended in 1: 10 guinea pig complement (B-D MCrieux, Marcy-l’Etoile, France) absorbed with 80 mg/ml agarose (Fluka AG, Buchs, Switzerland). After another incubation for 30 min at 37”C, cells were washed extensively with Hanks’ BSS. Activation of thywtus lymphocytes. Two procedures were employed to activate thymus cells from 5- to 6-wk-old CBA mice for C57BL/6 antigens. One involved ip injection of lo7 thymus cells into 850 R (C57BL/6 X CBA) Fl mice; activated cells were harvested from spleens 5 days later (4). The other procedure followed the slightly different protocol of Sprent and Miller (7) and was conducted by Dr. J. Sprent: lo8 cells were injected iv and 10s cells ip into 750 R (C57BL/6 x CBA) Fl mice. To obtain T.TDL, thoracic ducts were cannulated on day 4 and drainage was for 24 hr. Cells activated in this mamler were also harvested from spleens on day 5. Preparation of pure T cells. CBA spleen T cells were obtained by fractionation of spleen cells on columns prepared with polymethylmetaacrylic plastic beads with an average diameter of 250 p (Degalan V 26, Degussa, Wolfgang AG, Hannau a.M., FRG) following the procedure of Wigzell and Andersson (8). Extensively washed beads were first coated with normal mouse globulin and incubated overnight at 4°C. Excess globulin was then removed from the column by flushing and beads were coated with rabbit anti-mouse globulin antiserum. After an incubation at 4°C for 2 hr excess antiserum was washed out and spleen cells, suspended in Hanks’ BSS were filtered at a concentration of 40 X 106/ml. Induction of Fl hybrid sera. A standard dose of lo6 parental strain lymphoid cells was injected once ip into corresponding adult Fl hybrid hosts (Table 1).
strain cell inoculum
from
BALB/c spleen nu/nu spleen CBA spleen CBA TDL CBA bone marrow CBA native thymus CBA activated thumus (spleenb) CBA activated thymus (spleen=) ’ CBA T.TDL CBA anti-C57BL/6 spleen CBA spleen + anti-6 and C’ CBA spleen T cell@
Parental
X C57BL/6)F1
X CBA)Fl
(C57BL/6
(15) WV
(6)
(13)
(6) (6)
(5) (9)
(6)
(50)
i;;
1:2048 0 (test 1:1-1:512) 1:1024 1:1024 1:s 0 (test l:l-1:8) 1:1024 1: 1024 1:128/1:64e 1:64 0 (test 1: l-l : 32) 1:1024
PAR Titers” antibody
MOUSESERA
Antireceptor
TITERS OF F~HYBRID
(BALB/c
Hosts (No.1
1
a Serum dilution blocking PAR formation. b From spleens of 850 R (C57BL/6 X CBA)FI mice given lo7 cells 5 days previously (see Materials and Methods). c From spleens of 750 R (C57BL/6 X CBA)FI mice given 2 X lo8 cells 5 days previously (see Materials and Methods). d Fractionation according to Wigzell and Anderssop (8). a Two independently raised sera.
1 2' 3 4 5 6 7 8 9 10 11 12
Serum
TABLE
ANTIRECEPTORANTIBODYANDALLOANTIBODY
1:2.56 1:256 1:128 1:128 1:256 0 (test l:l-1:128) 1:128 1:128 1:32/1:32e 1:1024 1:128 0 (test 1: l-l : 256)
Alloantibody
ii
E z if4
3
ALLOANTIGEN
AND
T CELL
RECEPTOR
ANTIBODIES
425
Hosts were exsanguinated 2 wk later, serum pools prepared and sera were heatinactivated (56°C for 30 min) and sterilized by Millipore-filtration (2). Determination of antireceptor antibody and of alloantibody activities of Fl sera. Blocked and unblocked recognition of alloantigens was determined with the PAR assay (9, 10). Assessment of recognition involved cocultivation in Hanks’ BSS of lo6 parental strain spleen cells with lo6 Fl hybrid spleen cells under tissue culture conditions for 4 hr [BALE/c + (BALB/c X C57BL/6)Fl] or for 7 hr [CBA + (C57BL/6 x CBA)Fl]. To reveal anti-receptor antibody titers, parental strain spleen cells carrying appropriate receptors were first treated with Fl serum, then washed and were finally cultivated with untreated Fl target spleen cells, as described (1, 2). Alloantibody titers of Fl sera were determined similarly. Fl hybrid spleen cells, carrying appropriate alloantigens, were first treated with serum, then washed and were finally cultivated with untreated parental strain spleen cells (2, 3). All antisera were tested in two-fold dilution steps. Detailed procedures for treatment of supernatants and for PAR determinations are given elsewhere (9, 10). RESULTS Injections of parental strain lymphoid cell inocula, such as spleen or node cells, containing mixtures of T and B lymphocytes, into adult Fl hybrid hosts have been shown to result in formation of anti-receptor antibodies and of alloantibodies (2, 10). As shown in Table 1, inoculation of spleen cells from BALB/c or from CBA mice into corresponding Fl hosts led to the formation of both activities (Sera 1, 3). If, however, spleen cells from congenitally athymic nude mice were injected, formation of an anti-receptor activity was completely missing, whereas a normal alloantibody titer was obtained (Serum 2). The dependency of anti-receptor antibody formation on T cell receptors was also revealed when CBA cell inocula of normal or low T cell contents were used for injections of (C57BL/6 x CBA) Fl mice. CBA TDL (Serum 4) reacted similarly to CBA spleen cells, but CBA bone marrow cells (Serum 5) incited only a weak anti-receptor antibody response. Marrow cells, however, formed a normal alloantibody titer. While native thymus cells failed to respond to all (Serum 6), results identical to spleen cell inoculation attended the injection of CBA thymus cells activated for C57BL/6 transplantation antigens, provided activated thymus cells were taken from spleens of 850 R (C57BL/6 x CBA) Fl mice 5 days after injection of 10’ CBA thymus cells (Serum 7). A similar outcome was observed when a slightly different activation procedure (7) was employed (Serum S) . Considerably reduced titers for both activities were, on the other hand, found when similarly activated thymus cells (7) were employed, but harvested as thoracic duct small lymphocytes (T.TDL j (Serum 9). With respect to the anti-receptor activity, these cells elicited a response similar to that observed with CBA spleen cells sensitized to C57BL/6 alloantigens by skin graft rejection (Serum 10). On the other hand, there was a marked difference in alloantibody titer between Serum 9 (induced with T.TDL) and Serum 10 (induced with sensitized cells). The injection of CBA cell populations into (C57BL/6 X CBA) Fl hosts representing extremes with respect to T and B cell contents most beautifully illustrated their respective roles. Removal of T cells by anti-BCSH antiserum and complement resulted in a serum devoid of anti-receptor activity but displaying normal alloanti-
426
H.
RAMSEIER
body activity (Serum ll), whereas removal of B cells by column sieving resulted in a serum totally lacking alloantibody activity but showing normal anti-receptor antibody activity (Serum 12). DISCUSSION The object of this study was to establish that antibodies directed towards cellbound receptors for transplantation antigens were antibodies elicited by T cell receptors, and not by B cell receptors, The evidence obtained favors this interpretation. Injection of parental strain cell populations containing both T and B cells into Fl hosts invariably resulted in the elaboration of antireceptor antibodies and alloantibodies. (Table 1) (2, 10). Injections of cells lacking T or B cells, respectively, gave complementary results : cell suspensions devoid of T cells, such as spleen cells from nude mice or from normal mice depleted of T cells, failed to provoke formation of anti-receptor antibodies. The B cells of such inocula appeared to differentiate into antibody-forming cells, a process ultimately resulting in normal alloantibody titers. On the other hand, cell suspensions devoid of B cells, such as column-sieved spleen cells, could not form alloantibodies, but their T cells incited formation of normal anti-receptor antibody activity. Cell inocula consisting of intermediate proportions of T and B cells, such as spleen cells and TDL, incited and formed both serum activities, but two-fold serum dilutions failed to reveal differences corresponding to the known proportions of T and B cells (11). Injection of bone marrow cells, reported to contain only few antigen-sensitive T cells (12, 13), however, disclosed quantitative aspects : a small titer of only 1: 8 anti-receptor antibody was induced. Obviously, these cells appeared to contain high numbers of B cell precursors, as documented by the alloantibody titer they were capable of elaborating. Native thymus cells (106, but also 107- not shown) did not reveal sufficient numbers of T cells with immunogenic receptors or B cell precursors capable of differentiating under the experimental conditions used; results similar to those obtained previously with thymus cells from A strain mice (4). Activated thymus cells, on the other hand, responded. While the procedures employed to activate thymus lymphocytes for alloantigens (4, 7) appeared to have no influence on ultimate antireceptor antibody and alloantibody titers, the organ from which activated cells were taken did. Activated cells harvested from spleens of lethally irradiated Fl hosts behaved like spleen cells or TDL from normal mice, but when harvested from lymph (T.TDL), they behaved like sensitized cells with respect to T but not to B cells. As has been found also with rat cells (Ramseier, unpublished) sensitized cells incited considerably lower titers of antireceptor antibodies, presumably because of their cytotoxicity (14), which is likely to be directed against the very cell population that produced anti-receptor antibodies, i.e., Fl lymphoid cells. Together with evidence obtained from cytotoxic tests (15, 16), similarity in antireceptor antibody titers of sensitized cells and of T.TDL suggests common properties. Identical behavior for these cells has also been found for kinetic aspects of PAR formation (5). On the basis of alloantibody titers found for sensitized spleen cells and for T.TDL (serum 9, lo), marked differences in their respective B cell contents can be deduced. Spleens of sensitized mice already possess alloantibody-forming B cells and their injection into Fl hosts simply led to increased production. T.TDL, on the
ALLOANTIGEN
AND
T CELL
RECEPTOR
ANTIBODIES
427
other hand, have been reported to contain less than 1% B cells (17). Low alloantibody titers were, therefore, expected and only 1: 32 were found. In fair agreement with the results of Sprent and Miller (17)) this titer corresponds to that formed by lo3 CBA spleen cells (or 600 CBA B spleen cells), as has been revealed by critical dilution experiments (Ramseier, unpublished). The reason(s) why spleen and thoracic duct as sources of activated thymus cells differ so widely with respect to the putative immune status of harvested cells is not known, The observations merely point to profound qualitative differences. The immune status of activated thymus cells taken from spleens as reported here, is different from that based on cytotoxic tests (6, 18). In these similarly activated cells were cytotoxic, a property assigned only to T.TDL (16, 19) and to cells from sensitized mice. A possible reason for this discrepancy might be quantitative; up to 20-fold higher numbers of cells were used in cytotoxic tests. Results obtained with inocula free of T cells would indicate that alloantibody formation might be independent of T cell cooperation. However, even such inocula might have enlisted cooperative influences in intact Fl hosts (and as has been shown with spleen cells from nude mice even in lethally irradiated Fl hosts, Ramseier, unpublished). In view of recent results of Katz et a,l. (20) semiallogeneic T-B cell cooperation appears to be a real possibility. Identification of anti-receptor antibodies, or anti-RS antibodies as they have been called elsewhere (2, lo), as anti-T cell receptor antibodies is particularly satisfactory in view of their high specificity (2, 10, 21). It might be concluded that, at least for alloantigens, receptor specificity of T cells is as diversified as has been reported for B cell receptors (11). Similar degrees of specificity of T and B cell receptors for alloantigens might have been anticipated on the basis of findings’ indicating close antigenic association (if not identity) of B cell products (idiotype or aliotype of alloantibodies) and cellular receptors both under ifz witro (22, 23) and in viva (24, 25) conditions. ACKNOWLEDGMENTS I thank Prof. J. Lindenmann for stimulating discussions, Dr. H. Binz for help with the column technique and Miss D. Pliiss and Miss R. Leemann for expert technical assistance.
REFERENCES 1. Ramseier, H., and Lindenmann, J., Eur. J. Immunol. 1, 348, 1971. 2. Ramseier, H., and Lindenmann, J., Transplant. Rev. 10, 57, 1972. 3. Ramseier, H., and Lindenmann, J., Eur. J. Immunol. 1, 441, 1971. 4. Ramseier, H., Cell. Immunol. 8, 177, 1973. 5. Ramseier, H., Nnfure (hdolz) 246, 351, 1973. 6. Cerottini, J. C., Nordin, A. A., and Brunner, K. T., Nafztre (Londojc) 228, 1308, 1970. 7. Sprent, J., and Miller, J. F. A. P., Ccl/. Znzm~nol. 3, 361, 1972. 8. Wigzell, H., and Andersson, B., J. Exp. Med. 129, 23, 1969. 9. Ramseier, H., J. Exp. Med. 130, 1279, 1969. 10. Ramseier, H., Curr. Topics Microbial. Immzmol. 60, 31, 1973. 11. Katz, D. H., and Benacerraf, B., Advan. Imnurcol. 15,2, 1972. 12. Miller, J. F. A. P., Basten, A., Sprent, J., and Cheers, C., Cell. Immtlnol. 2, 469, 1971. 13. Raff, M. C., Nase, S., and Mitchison, N., Nature (Lo~dorz) 230, 50, 1971. 14. Wilson, D. B., and Billingham, R. E., Advan. Imm~~~tol.7, 189, 1967. 15. Cerottini, J. C., Nordin, A. A., and Brunner, K. T., J. Exp. Med. 134, 553, 1971. 16. Sprent, J., and Miller, J. F. A. P., Nature New Biol. 234, 195, 1971.
428
H.
RAMSEIER
17. Sprent, J., and Miller, J. F. A. P., Cell. Zmmunol. 3, 385, 1972. 18. Golstein, P., Blomgren, H., and Svedmyr, E. A. J., Cell. Immunol. 7, 213, 1973. 19. Sprent, J., and Miller, J. F. A. P., Cell. Immunol. 3, 213, 1972. 20. Katz, D. H., Hamaoka, T., and Benacerraf, B., J. Exp. Med. 137, 1405, 1973. 21. Ramseier, H., Eur. J. Immunol. 3, 164, 1973. 22. Ramseier, H., and Lindenmann, J., J. E.rp. Med. 134, 1083, 1971. 23. Binz, H., and Lindenmann, J., J. Exp. Med. 136, 872, 1972. 24. Binz, H., and Lindenmann, J., Stand. J. Zmmuwol. 1, 339, 1972. 246, 146, 1973. 2.5. Binz, H., Wigzell, H., and Lindenmann, J., Nafwe (Loadox)
12,422-428 (1974)
IMMUNOLOGY
Antibodies
to T Cell Receptors and to Histocompatibility
Antigens1
H. RAMSEIER Division
of Experimental Microbiology, Microbiology, University Received
Departme& of Zurich
of Medical
November 14, 1973
The injection of 10’ parental strain spleen cells into adult Fl hybrid mice resulted in the formation of two serum activities: anti-receptor antibodies and alloantibodies. To demonstrate the respective roles of T and B cells in the elicitation of these serum activities, parental strain lymphoid cell suspensions containing varying proportions of T and B cells, or consisting only of T or B cells, were employed as inocula for. Fl mice. The results indicated that the injection of pure T cells led to the formation of antireceptor antibodies only, while the injection of pure B cells resulted exclusively in formation of alloantibodies, suggesting that anti-receptor antibodies were structures elicited by T cell receptors. Alloantibodies appear to be formed as a consequence of the interaction of B cell receptors with alloantigen.
INTRODUCTION The reaction between receptors of lymphoid cells and histocompatibility antigens is highly specific. It can be interfered with by two equally specific manipulations: blocking of receptors by anti-receptor antisera (1, 2) or blocking of transplantation antigens by alloantisera (2, 3). It is known that the injection of lymphoid cells from spleens or nodes of normal (unsensitized) animals of parental origin into appropriate adult Fl hybrid hosts results in the formation within the same serum of both anti-receptor antibodies and of alloantibodies (2). While it appears likely that alloantibodies are ultimately formed by bone marrow-derived (B) cells, it is not certain whether anti-receptor antibodies are formed in response to receptors present on thymus-derived (T) cells or to those present on B cells. Two observations make it likely that anti-receptor antibodies are directed against T cell receptors. First, thymus lymphocytes activated for alloantigens by in viva passage through irradiated recipients represent high numbers of antigen-sensitive T cells; they also elicit formation of anti-receptor antibodies (4). Second, in the test system employed to measure antigenic recognition, the PAR assay, only T cells, but not B cells, are essential (5). To establish the critical roles of T and B cells in the initiation of anti-receptor antibodies and of alloantibodies, a study was undertaken in which lymphoid cells 1 Abbreviations used in this paper : B cells, bone marrow-derived cells; BBS, balanced salt solution ; PAR, product of antigenic recognition; T cells, thymus-derived cells; TDL, thoracic duct small lymphocytes ; T.TNDL, thymus cells activated for alloantigens and harvested as small lymphocytes from thoracic duct. 422 CopyrightQ All
rights
1974 by of reproduction
AcademicPress, Inc. in any
form
reserved.
ALLOANTIGEK
of different origins raise Fl sera. The clusively against T as a consequence of
Am
T CELL RECEPTOR ANTIRODIES
423
and with varying contents of T and B cells were employed to results indicated that anti-receptor antisera were directed excell receptors, whereas alloantibodies appeared to be formed the interaction of B cell receptors with transplantation antigens. MATERIALS
AND
METHODS
Anbmls and lymphoid cells. Mice of inbred strains CBA, BALB/c and Fl hybrids of these strains with C57BL/6 were employed as donors of lymphoid cells. Nude mice (nu/nu)were kindly donated by Dr. B. Kindred; they were animals backcrossed to BALB/c mice for 7 generations. CBA thoracic duct small lymphocytes (TDL) and CBA thymus lymphocytes activated for C57BL/6 alloantigens and harvested from the thoracic duct (T.TDL) were gifts from Dr. J. Sprent. Other cells employed were from spleen, bone marrow and thymus. All cell suspensions were prepared in Hanks’ BSS (Gibco, New York, NY) without serum but fortified with 100 IU penicillin and SO pg/ml streptomycin. This medium was also used to maintain cells in short term cultures (PAR assay). Sensitization. To obtain cells actively sensitized to alloantigens, spleen cell suspensions were prepared from CBA mice 21 days after full-thickness C57BL/6 skin grafts. Treatment of spleen cells with anti-theta a,nttierunz and covtzplement. Anti-OC3H antiserum was prepared according to Cerottini et al. (6). Spleen cells at a concentration of 2 x lo7 were suspended in 1: 10 anti-BC3H and incubated for 30 min at 37°C under tissue culture conditions (5% CO2 in moist air). They were then centrifuged (130s for 8 min) and resuspended in 1: 10 guinea pig complement (B-D MCrieux, Marcy-l’Etoile, France) absorbed with 80 mg/ml agarose (Fluka AG, Buchs, Switzerland). After another incubation for 30 min at 37”C, cells were washed extensively with Hanks’ BSS. Activation of thywtus lymphocytes. Two procedures were employed to activate thymus cells from 5- to 6-wk-old CBA mice for C57BL/6 antigens. One involved ip injection of lo7 thymus cells into 850 R (C57BL/6 X CBA) Fl mice; activated cells were harvested from spleens 5 days later (4). The other procedure followed the slightly different protocol of Sprent and Miller (7) and was conducted by Dr. J. Sprent: lo8 cells were injected iv and 10s cells ip into 750 R (C57BL/6 x CBA) Fl mice. To obtain T.TDL, thoracic ducts were cannulated on day 4 and drainage was for 24 hr. Cells activated in this mamler were also harvested from spleens on day 5. Preparation of pure T cells. CBA spleen T cells were obtained by fractionation of spleen cells on columns prepared with polymethylmetaacrylic plastic beads with an average diameter of 250 p (Degalan V 26, Degussa, Wolfgang AG, Hannau a.M., FRG) following the procedure of Wigzell and Andersson (8). Extensively washed beads were first coated with normal mouse globulin and incubated overnight at 4°C. Excess globulin was then removed from the column by flushing and beads were coated with rabbit anti-mouse globulin antiserum. After an incubation at 4°C for 2 hr excess antiserum was washed out and spleen cells, suspended in Hanks’ BSS were filtered at a concentration of 40 X 106/ml. Induction of Fl hybrid sera. A standard dose of lo6 parental strain lymphoid cells was injected once ip into corresponding adult Fl hybrid hosts (Table 1).
strain cell inoculum
from
BALB/c spleen nu/nu spleen CBA spleen CBA TDL CBA bone marrow CBA native thymus CBA activated thumus (spleenb) CBA activated thymus (spleen=) ’ CBA T.TDL CBA anti-C57BL/6 spleen CBA spleen + anti-6 and C’ CBA spleen T cell@
Parental
X C57BL/6)F1
X CBA)Fl
(C57BL/6
(15) WV
(6)
(13)
(6) (6)
(5) (9)
(6)
(50)
i;;
1:2048 0 (test 1:1-1:512) 1:1024 1:1024 1:s 0 (test l:l-1:8) 1:1024 1: 1024 1:128/1:64e 1:64 0 (test 1: l-l : 32) 1:1024
PAR Titers” antibody
MOUSESERA
Antireceptor
TITERS OF F~HYBRID
(BALB/c
Hosts (No.1
1
a Serum dilution blocking PAR formation. b From spleens of 850 R (C57BL/6 X CBA)FI mice given lo7 cells 5 days previously (see Materials and Methods). c From spleens of 750 R (C57BL/6 X CBA)FI mice given 2 X lo8 cells 5 days previously (see Materials and Methods). d Fractionation according to Wigzell and Anderssop (8). a Two independently raised sera.
1 2' 3 4 5 6 7 8 9 10 11 12
Serum
TABLE
ANTIRECEPTORANTIBODYANDALLOANTIBODY
1:2.56 1:256 1:128 1:128 1:256 0 (test l:l-1:128) 1:128 1:128 1:32/1:32e 1:1024 1:128 0 (test 1: l-l : 256)
Alloantibody
ii
E z if4
3
ALLOANTIGEN
AND
T CELL
RECEPTOR
ANTIBODIES
425
Hosts were exsanguinated 2 wk later, serum pools prepared and sera were heatinactivated (56°C for 30 min) and sterilized by Millipore-filtration (2). Determination of antireceptor antibody and of alloantibody activities of Fl sera. Blocked and unblocked recognition of alloantigens was determined with the PAR assay (9, 10). Assessment of recognition involved cocultivation in Hanks’ BSS of lo6 parental strain spleen cells with lo6 Fl hybrid spleen cells under tissue culture conditions for 4 hr [BALE/c + (BALB/c X C57BL/6)Fl] or for 7 hr [CBA + (C57BL/6 x CBA)Fl]. To reveal anti-receptor antibody titers, parental strain spleen cells carrying appropriate receptors were first treated with Fl serum, then washed and were finally cultivated with untreated Fl target spleen cells, as described (1, 2). Alloantibody titers of Fl sera were determined similarly. Fl hybrid spleen cells, carrying appropriate alloantigens, were first treated with serum, then washed and were finally cultivated with untreated parental strain spleen cells (2, 3). All antisera were tested in two-fold dilution steps. Detailed procedures for treatment of supernatants and for PAR determinations are given elsewhere (9, 10). RESULTS Injections of parental strain lymphoid cell inocula, such as spleen or node cells, containing mixtures of T and B lymphocytes, into adult Fl hybrid hosts have been shown to result in formation of anti-receptor antibodies and of alloantibodies (2, 10). As shown in Table 1, inoculation of spleen cells from BALB/c or from CBA mice into corresponding Fl hosts led to the formation of both activities (Sera 1, 3). If, however, spleen cells from congenitally athymic nude mice were injected, formation of an anti-receptor activity was completely missing, whereas a normal alloantibody titer was obtained (Serum 2). The dependency of anti-receptor antibody formation on T cell receptors was also revealed when CBA cell inocula of normal or low T cell contents were used for injections of (C57BL/6 x CBA) Fl mice. CBA TDL (Serum 4) reacted similarly to CBA spleen cells, but CBA bone marrow cells (Serum 5) incited only a weak anti-receptor antibody response. Marrow cells, however, formed a normal alloantibody titer. While native thymus cells failed to respond to all (Serum 6), results identical to spleen cell inoculation attended the injection of CBA thymus cells activated for C57BL/6 transplantation antigens, provided activated thymus cells were taken from spleens of 850 R (C57BL/6 x CBA) Fl mice 5 days after injection of 10’ CBA thymus cells (Serum 7). A similar outcome was observed when a slightly different activation procedure (7) was employed (Serum S) . Considerably reduced titers for both activities were, on the other hand, found when similarly activated thymus cells (7) were employed, but harvested as thoracic duct small lymphocytes (T.TDL j (Serum 9). With respect to the anti-receptor activity, these cells elicited a response similar to that observed with CBA spleen cells sensitized to C57BL/6 alloantigens by skin graft rejection (Serum 10). On the other hand, there was a marked difference in alloantibody titer between Serum 9 (induced with T.TDL) and Serum 10 (induced with sensitized cells). The injection of CBA cell populations into (C57BL/6 X CBA) Fl hosts representing extremes with respect to T and B cell contents most beautifully illustrated their respective roles. Removal of T cells by anti-BCSH antiserum and complement resulted in a serum devoid of anti-receptor activity but displaying normal alloanti-
426
H.
RAMSEIER
body activity (Serum ll), whereas removal of B cells by column sieving resulted in a serum totally lacking alloantibody activity but showing normal anti-receptor antibody activity (Serum 12). DISCUSSION The object of this study was to establish that antibodies directed towards cellbound receptors for transplantation antigens were antibodies elicited by T cell receptors, and not by B cell receptors, The evidence obtained favors this interpretation. Injection of parental strain cell populations containing both T and B cells into Fl hosts invariably resulted in the elaboration of antireceptor antibodies and alloantibodies. (Table 1) (2, 10). Injections of cells lacking T or B cells, respectively, gave complementary results : cell suspensions devoid of T cells, such as spleen cells from nude mice or from normal mice depleted of T cells, failed to provoke formation of anti-receptor antibodies. The B cells of such inocula appeared to differentiate into antibody-forming cells, a process ultimately resulting in normal alloantibody titers. On the other hand, cell suspensions devoid of B cells, such as column-sieved spleen cells, could not form alloantibodies, but their T cells incited formation of normal anti-receptor antibody activity. Cell inocula consisting of intermediate proportions of T and B cells, such as spleen cells and TDL, incited and formed both serum activities, but two-fold serum dilutions failed to reveal differences corresponding to the known proportions of T and B cells (11). Injection of bone marrow cells, reported to contain only few antigen-sensitive T cells (12, 13), however, disclosed quantitative aspects : a small titer of only 1: 8 anti-receptor antibody was induced. Obviously, these cells appeared to contain high numbers of B cell precursors, as documented by the alloantibody titer they were capable of elaborating. Native thymus cells (106, but also 107- not shown) did not reveal sufficient numbers of T cells with immunogenic receptors or B cell precursors capable of differentiating under the experimental conditions used; results similar to those obtained previously with thymus cells from A strain mice (4). Activated thymus cells, on the other hand, responded. While the procedures employed to activate thymus lymphocytes for alloantigens (4, 7) appeared to have no influence on ultimate antireceptor antibody and alloantibody titers, the organ from which activated cells were taken did. Activated cells harvested from spleens of lethally irradiated Fl hosts behaved like spleen cells or TDL from normal mice, but when harvested from lymph (T.TDL), they behaved like sensitized cells with respect to T but not to B cells. As has been found also with rat cells (Ramseier, unpublished) sensitized cells incited considerably lower titers of antireceptor antibodies, presumably because of their cytotoxicity (14), which is likely to be directed against the very cell population that produced anti-receptor antibodies, i.e., Fl lymphoid cells. Together with evidence obtained from cytotoxic tests (15, 16), similarity in antireceptor antibody titers of sensitized cells and of T.TDL suggests common properties. Identical behavior for these cells has also been found for kinetic aspects of PAR formation (5). On the basis of alloantibody titers found for sensitized spleen cells and for T.TDL (serum 9, lo), marked differences in their respective B cell contents can be deduced. Spleens of sensitized mice already possess alloantibody-forming B cells and their injection into Fl hosts simply led to increased production. T.TDL, on the
ALLOANTIGEN
AND
T CELL
RECEPTOR
ANTIBODIES
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other hand, have been reported to contain less than 1% B cells (17). Low alloantibody titers were, therefore, expected and only 1: 32 were found. In fair agreement with the results of Sprent and Miller (17)) this titer corresponds to that formed by lo3 CBA spleen cells (or 600 CBA B spleen cells), as has been revealed by critical dilution experiments (Ramseier, unpublished). The reason(s) why spleen and thoracic duct as sources of activated thymus cells differ so widely with respect to the putative immune status of harvested cells is not known, The observations merely point to profound qualitative differences. The immune status of activated thymus cells taken from spleens as reported here, is different from that based on cytotoxic tests (6, 18). In these similarly activated cells were cytotoxic, a property assigned only to T.TDL (16, 19) and to cells from sensitized mice. A possible reason for this discrepancy might be quantitative; up to 20-fold higher numbers of cells were used in cytotoxic tests. Results obtained with inocula free of T cells would indicate that alloantibody formation might be independent of T cell cooperation. However, even such inocula might have enlisted cooperative influences in intact Fl hosts (and as has been shown with spleen cells from nude mice even in lethally irradiated Fl hosts, Ramseier, unpublished). In view of recent results of Katz et a,l. (20) semiallogeneic T-B cell cooperation appears to be a real possibility. Identification of anti-receptor antibodies, or anti-RS antibodies as they have been called elsewhere (2, lo), as anti-T cell receptor antibodies is particularly satisfactory in view of their high specificity (2, 10, 21). It might be concluded that, at least for alloantigens, receptor specificity of T cells is as diversified as has been reported for B cell receptors (11). Similar degrees of specificity of T and B cell receptors for alloantigens might have been anticipated on the basis of findings’ indicating close antigenic association (if not identity) of B cell products (idiotype or aliotype of alloantibodies) and cellular receptors both under ifz witro (22, 23) and in viva (24, 25) conditions. ACKNOWLEDGMENTS I thank Prof. J. Lindenmann for stimulating discussions, Dr. H. Binz for help with the column technique and Miss D. Pliiss and Miss R. Leemann for expert technical assistance.
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17. Sprent, J., and Miller, J. F. A. P., Cell. Zmmunol. 3, 385, 1972. 18. Golstein, P., Blomgren, H., and Svedmyr, E. A. J., Cell. Immunol. 7, 213, 1973. 19. Sprent, J., and Miller, J. F. A. P., Cell. Immunol. 3, 213, 1972. 20. Katz, D. H., Hamaoka, T., and Benacerraf, B., J. Exp. Med. 137, 1405, 1973. 21. Ramseier, H., Eur. J. Immunol. 3, 164, 1973. 22. Ramseier, H., and Lindenmann, J., J. E.rp. Med. 134, 1083, 1971. 23. Binz, H., and Lindenmann, J., J. Exp. Med. 136, 872, 1972. 24. Binz, H., and Lindenmann, J., Stand. J. Zmmuwol. 1, 339, 1972. 246, 146, 1973. 2.5. Binz, H., Wigzell, H., and Lindenmann, J., Nafwe (Loadox)