Tissue Antigens (1977),9, 11-16 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s)
Lymphoblastoid Cell Lines of Homozygous Typing Cells Used for Sensitization in PLT Nancy Reinsmoen, Edmond J. Yunis, Fritz H. Bach and Marilyn L. Bach Department of Laboratory Medicine and Pathology University of Minnesota Medical School Minneapolis, Minnesota, U .S .A. and The Immunobiology Research Center and Departments of Medical Genetics, Surgery and Pediatrics University of Wisconsin Madison, Wisconsin, U.S.A.
Lymphoblastoid cell lines of homozygous typing cells were used as the sensitizing cells in MLC t o prepare PLT cells. Results obtained using such cells against a panel of restimulating cells were compared t o those obtained using regular PLT cells in which priming had been accomplished with normal peripheral blood lymphocytes. It appears that lymphoblastoid cell lines can be used for this purpose; the advantages of such an approach are given. Received for publication 8 June, accepted 5 August 1976
The primed lymphocyte typing (PLT) test was introduced (Bach et al. 1975, Sheehy et al. 1975,Sonde1 et al. 1976) as a method for HLA-D typing to complement the homozygous typing cell approach (Bradley et al. 1972,Thorsby & Piazza 1975). The PLT method is based on observations in mouse (Andersson & Hayry 1973, Mac-
Donald et al. 1974,Alter et al. 1976) and man (Fradelizi & Dausset 1975, Svedmyr 1975, Zier & Bach 1975) that secondarytype proliferative responses can be generated in vitro. Potential advantages of the PLT method (in which lymphocytes of one individual are “sensitized” in MLC to HLA one haplotype different stimulating cells
This work is supported in part by PHS grant HL-06314 (EJY) and NIH grants AI-00576, AI-08439, CA-16836 and National Foundation-March of Dimes grants CRBS 246 and 6-76-213 (FHB & MLB). This is paper no. 2002 from the Laboratory of Genetics and paper no. 8 7 from the Immunobiology Research Center, The University of Wisconsin, Madison, Wisconsin 53706.
12 NANCY REINSMOEN, EDMOND J . YUNIS, FRITZ H. BACH AND MARILYN L. BACH
and subsequently used as test reagents) are the following. First, PLT cells can be made against essentially any HLA-D haplotype. Second, the test results are available within a day when needed. Third, since it appears that a single D haplotype codes for several determinants (Dupont et al. 1973, Bach et al. 1975), it may be possible to prepare PLT cells against single HLA-D determinants depending on the HLA-D phenotype of responder and stimulator in the primary MLC. It would be of obvious value to have a constant and readily available source of stimulating cells for MLC sensitization that carry a known D locus determinant. Eventually such stimulator cells could be used to sensitize responding cells that lack that determinant but have all other D determinants of the stimulating cell. To that end, we have used lymphoblastoid cell lines from homozygous typing cells as the sensitizing cells in MLC. We have then restimulated the resulting PLT cells with normal peripheral blood lymphocytes from the donor of the lymphoblastoid cell line, other normal lymphocytes carrying the same HLA-D region antigents, as well as cells not carrying those antigens. The results provide preliminary evidence that lymphoblastoid cell lines, which appear to carry the HLA-D determinants (Netzel et al. 1975, Svedmyr et al. 1975), can be used for PLT priming. Materials and methods PLT cells are generated by the method recently described (Sheehy & Bach 1976). In brief, 1 x lo7 responding cells, which are negative for a given DW cluster, were sensitized in separate flasks to either 1 x lo7 normal lymphocytes of the homozygous typing cell donor or 1 x lo6 lymphoblastoid cells from that homozygous typing cell. The primed cells were harvested 10
days later and used for the experiment. PLT cultures were prepared in round bottom wells with 5 x lo4 responding cells and 5 x lo4restimulating cells. The cultures were labeled for 1 6 h amd precipitated (O’Leary et al. 1976). Restimulating cells were used from the original donor of the homozygous typing cell (reference restimulating cells); from cells of the original responding cell donor (control restimulating cells); and from various other individuals (test restimulating cells). Test cells were classified as either carrying the same HLA-D haplotype as the reference cell on the basis of genotyping within a family or, when test cells were used which were unrelated to the control and reference cell, by homozygous typing cell testing.
Results Shown in Figures 1A and 1B are PLT results with two different PLT cells. Results in Figure 1A are obtained using a PLT cell in which cells of a responder that did not carry a given HLA-D cluster were sensitized to the peripheral blood lymphocytes of a homozygous typing cell donor (KH) (Furcht et al. 1976). (This homozygous typing cell does not show a significant association with the presently defined DW clusters.) The PLT cell was restimulated with the normal lymphocytes of the reference cell donor (KH), three family members carrying the same D haplotype (individuals E, F and G), a test cell from an unrelated individual carrying the same D haplotype, by homozygous typing cell testing (individual H), and cells of four individuals (I, J , K and L) not carrying the D cluster by testing with the homozygous cell. The results shown in Figure 1 B are obtained using a PLT cell in which cells from a lymphoblastoid cell line from cells of KH were used for sensitization in the
SENSITIZATION IN PLT 11
13
10
PLT s*nmlmad to
PLT sans811zad L O normal
pmriphmral blood Imuhocyles
lymphoblastoid cell line
n I
: X
t
1
2 Day 01 labding
3 Day of labeling
Figure 1 . Results of PLT studies using PLT cells sensitized t o either normal perpipheral blood leukocytes or the lymphoblastoid cell lines of a homozygous typing cell donor. Cultures were labeled at 24, 4 8 and 72 hours with tritiated thymidine and harvested 16 hours later. The dotted line represents the results obtained with the reference cell, the dashed line the results obtained with test cells carrying the sensitizing DW haplotype and the solid line results obtained with test Celts not carrying the sensitizing DW haplotype. The responding cell donor for experiments shown in 1A and 1B has the HLA phenotype AW23, AW31, B7 in a primary MLC gave a relative response t o KH stimulating cells of 60%. The HLA phenotypes of the eight test restimulating cells are as follows (with the relative response to stimulating cells of KH given in parentheses): E, AW19, BS, BW16 (13); F,AW24, AW31, 8 5 (11); G , AW23, AW31, B5 (13); H, AW31, B5 ( 0 ) ; I, AW19, BW15, BW22 (93); J, A l , A 3 , B 8 , B14, DW3 (69); L, A l , A3, B7, BW15, BW2 (76); K, A3, AW19, B14, B27, DW2 (74). The homozygous typing cell KH is AW24, AW31, BS. The responder for the experiment shown in Figures 1C and I D is AW19, BW1S and BW16 (70) and shows a relative response t o the homozygous stimulating cell, MS, of 76%. The HLA phenotypes of the individuals used in that experiment are as follows (with the relative response t o the homozygous typing cell given in parentheses): M, A l , A3, B7, BW15, DW2 (23); N, A3, AW19, B14, B27, DW2 (21); 0, A2, A3, 8 5 , B7, DW2 (19); P, A2, B7, BW17, DW2 (33);Q, A2, B12, BWlS, DW4 (81); R, A9, A l l , B18, B14 (76); S, A l , A9, B5. BW22 (77); T, A2, AW19, B12, 88 (43). The homozygous typing cell (MS) is A2, A3.87, DW2.
14
NANCY REINSMOEN, EDMOND J. YUNIS, FRITZ H. BACH AND MARILYN L. BACH It
1D
PLT Sensiltzed 10 normal
PLT
1
15
peripheral blood IeukocYIes
sensilired l o Iymphoblasloid c e l l line
-
2
3
Day 01 labeling
primary MLC. Otherwise, the resulting PLT cells were handled identically. The results shown in Figures 1C and 1D are from an experiment in which primary MLC sensitization was accomplished with peripheral blood lymphocytes of a DW2 homozygous typing cell (MS) (Reinsmoen et al. 1975) (Figure 1C) and with the lymphoblastoid cell line of that homozygous typing cell (Figure 1D). Restirnulation of these PLT cells was done with normal peripheral blood lymphocytes of the reference cell donor, four unrelated individuals (M, N , 0 and P) whose cells are positive for DW2 by homozygous typing cell testing) and four unrelated test cells (individuals Q, R , S and T) whose cells are negative for DW2.
Day of Isbeling
Discussion Results obtained with PLT cells appear to measure antigens determined by the HLAD region in that results of PLT tests in unrelated individuals are predictive of primary MLC reactions (Sheehy et al. 1975) and in HLA-B - HLA-D recombinant individuals it is the D region that is important in restimulation (Mawas et al. 1975, Bradley et al. 1976). Whereas one can “type” between 2 x lo3 and 5 x lo3 test cells with PLT cells generated from one pint of blood, it will be of obvious advantage to use a sensitizing cell in the form of a lymphoblastoid cell line for the reasons given in the introduction. Our results, while preliminary, suggest that lymphoblastoid cell lines can be used in
15
SENSITIZATION IN PLT
this way. Lymphoblastoid cell line cells are autostimulatory in the primary MLC (Green & Sell 1970, Knight et al. 1971), however, since we have used normal lymphocytes for the restimulating cell for the PLT test, the autostimulatory effect of the lymphoblastoid cen’ line is probably not pertinent to the results obtained. The normal low level “response” when control restimulating cells are used is consistent with this conclusion. We usually assay response of PLT cells prior to 40 o r 48 h at the latest (Bach et al. 1975, Sheehy et al. 1975, Sheehy & Bach 1976, Sondel et al. 1976), i.e. prior to the appearance of secondary inhibitory effects (as can, for instance, be seen with restimulating cells E and F in Figure 1A). We would, thus, make our comparison of the data obtained in these experiments when the PLT cultures were labeled at 2 4 h for an additional 1 6 h . The possibly greater thymidine incorporation seen in PLT cells prepared against lymphoblastoid cell lines needs further confirmation but may be a useful aspect of this approach. In the experiments shown in Figure 1 as in other experiments we have performed to date, the reference restimulating cell gives maximal or near maximal restimulation, and other test restimulating cells that carry the HLA-D haplotype of the reference cell on the average give higher restimulation that test cells that d o not carry that D haplotype. The findings in these experiments that PLT cells made against lymphoblastoid cell lines give very similar results to those sensitized to normal cells, supports this approach for preparing PLT cells. I t has recently been demonstrated that lymphocytes may be primed against the surface antigens presented o n leukemic cells. A secondary proliferative response can be produced by the leukemic cells which appears to be independent of the
HLA-D determinants of the priming cell (Reinsmoen et al. 1976). These priming and restimulatory antigens are thought to be those that are stimulatory in an MLC (Bach et al. 1969).
Acknowledgements We wish t o thank Drs. Wolfgang Leibold and Richard A. Gatti for establishing the lymphoblastoid cell lines from homozygous typing cells which were from the laboratory of Dr. E.J. Yunis at the University of Minnesota.
References Alter, B.J., GrillotCourvalin. C., Zier, K.S., Sondel, P.M. & Bach, F.H. (1976) Secondary CML: Importance of H-2 LD and S D factors. J . Exp. Med. 143, 1005-1014. Anderson. L.C. & Hayry, P. (1973) Specific priming of mouse thymusdependent lymphocytes to allogeneic cells in vitro. Eur. J. Immunol. 3, 595-599. Bach, M.L.,Bach, F.H. & Joo,P. (1960) Leukemiaassociated antigens in the mixed leukocyte culture test. Science 166, 1520-1522. Bach, F.H., Sondel, P.M.,Sheehy. M.J., Wank, K., Alter, B.J. & Bach, M.L. (1975) The complexity of the HL-A LD system: A PLT analysis, Histocompatibility Testing 1975, ed. Kissmeyer-Nielsen, F., p. 576-580. Munksgaard, Copenhagen. Bradley, B.A., Edwards, J.M., Dunn, D.C. & Calne, R.Y. (1972) Quantitation of mixed lymphocyte reaction by gene dosage phenornenon. Nature New Biology 240, 54-56. Bradley, B.A., Sheehy, M.J., Keuning, J . J . , Termijtelen, A., Franks, D. & van Kood, J.J. (1976) The phenotyping of HLA-D region products by negative and .positive mixed lymphocyte reactions. lmmunogenetics (in press). Dupont, B . , Jersild, C., Hansen, G.S., StaubNielsen, L., Thomsen, M. & Svejgaard, A. (1973) Multiple MLC (LD) determinants o n the same HL-A haplotype. Transplant. Proc. V , 1481-1487. Fradelizi, D. & Dausset, J . (1975) Mixed lympho-
16
NANCY REINSMOEN, EDMOND J. YUNIS, FRITZ H. BACH AND MARILYN L. BACH
of several MLR-S (MLC) homozygous cells and the typing for these determinants in the North American Caucasian population (Minnesota). Histocompatibility Testing 1 9 7 5 , ed. Kissmeyer-Nielsen, F., p. 459-463. Munksgaard, Copenhagen. Sheehy, M.J. & Bach, F.H. (1976)Primed LD typing (PLT) - technical considerations. Tissue Antigens (in press). Sheehy, M.J., Sondel, P.M., Bach, M.L., Wank. R. & Bach. F.H. (1975) HL-A LD (lymphocyte defined) typing: A rapid assay with primed 170,989-990. lymphocytes. Science 188,1308-1310. Knight, S.C., Moore, G.E. & Clarkson, B.D. (1971)Stimulation of autochthonous lympho- Sondel, P.M., Sheehy, M.J., Benike, C., Alter, B.J. & Bach. F.H. (1976)Specific detection cytes by cells from normal and leukaemic of HLA-D antigens using primed LD typing lines. Nature New Biol. 229,175-187. (PLT). Transplant. Proc. (in press). MacDonald, H.R., Engers, H.D., Cerottini, J.C. & Brunner, K.T. (1974) Generation of cyto- Svedmyr, E. (1975) Long-term maintenance in vitro of human T cells b y repeated exposure toxic T lymphocytes in vitro. 11. Effect of to the same stimulator cells. Scand. J . repeated exposure to alloantigens on t h e Immunol. 4,421-427. cytotoxic activity of long-term mixed leukocyte cultures. J . Exp. Med. 140. 718-730. Svedmyr. E., Leibold, W. & Gatti, R.A. (1975) Possible use of established cell lines for MLR Mawas, C.E.,Charmot, D., & Sasportes, M. (1975) locus typing. Tissue Antigens 5, 186-195. Secondary response of in vitro primed human lymphocytes to allogeneic cells. 1. Role of Thorsby, E. & Piazza, A. (eds.). (1975) Joint HL-A antigens and mixed lymphocyte reaction report from t h e sixth international histostimulating determinants in secondary in compatibility workshop conference. 11. Typing vitro proliferative response. Immunogenetics for HLA-D (LD-1 or MLC) determinants. 2,449-463. Histocompatibility Testing 1975, ed. KissNetzel, B., Mempel, W.,Albert, E.D., Baumann, P. meyer-Nielsen, F.. p. 414-458. Munksgaard, Copenhagen. & Grosse-Wilde, H. (1975) LD typing in lymphoblastoid cell lines. Immunogenetics 2 , Zier, K.S. & Bach, F.H. (1975) Secondary responses of human lymphocytes t o allo205-210. antigens in vitro. Scand. J. Immunol. 4, O’Leary, J., Reinsmoen, N. & Yunis, E.J. (1976) 607-61 1. Mixed lymphocyte reaction. Manual of Clinical Immunology (in press). Reinsmoen, N., Kersey, J. & Yunis, E.J. (1976) Address: Leukemia-associated antigens detected in N.Heinsmoen primed lymphocyte tests. Transplant. Proc. University of Minnesota (in press). Dept. of Laboratory Medicine and Pathology Reinsmoen, N., Stewart, M., Emme, L., Hanrahan, Medical School L.A., Dupont, B., Hansen, J.A., Friend, P., Box 198,Mayo Memorial Building Amos, D.B. & Yunis, E.J. (1975) Definition Minneapolis, Minnesota 55455
cyte reactivity of human lymphocytes primed in vitro. 1. Secondary response to allogenic lymphocytes. Eur. J . Immunol. 5, 295-301. Furcht, L.T., Chopyk, R.L., Yunis, E.J. & Greenberg, L.J. (1976)Relationship between immune responsiveness in vitro to streptococcal antigens and the HL-1 system in the Chippewa Indians. Fed. Proc 35, 353a. Green, S.S. & Sell, K.W. (1970)Mixed leukocyte stimulation of normal peripheral leukocytes by autologous lymphoblastoid cells. Science
Lymphoblastoid Cell Lines of Homozygous Typing Cells Used for Sensitization in PLT Nancy Reinsmoen, Edmond J. Yunis, Fritz H. Bach and Marilyn L. Bach Department of Laboratory Medicine and Pathology University of Minnesota Medical School Minneapolis, Minnesota, U .S .A. and The Immunobiology Research Center and Departments of Medical Genetics, Surgery and Pediatrics University of Wisconsin Madison, Wisconsin, U.S.A.
Lymphoblastoid cell lines of homozygous typing cells were used as the sensitizing cells in MLC t o prepare PLT cells. Results obtained using such cells against a panel of restimulating cells were compared t o those obtained using regular PLT cells in which priming had been accomplished with normal peripheral blood lymphocytes. It appears that lymphoblastoid cell lines can be used for this purpose; the advantages of such an approach are given. Received for publication 8 June, accepted 5 August 1976
The primed lymphocyte typing (PLT) test was introduced (Bach et al. 1975, Sheehy et al. 1975,Sonde1 et al. 1976) as a method for HLA-D typing to complement the homozygous typing cell approach (Bradley et al. 1972,Thorsby & Piazza 1975). The PLT method is based on observations in mouse (Andersson & Hayry 1973, Mac-
Donald et al. 1974,Alter et al. 1976) and man (Fradelizi & Dausset 1975, Svedmyr 1975, Zier & Bach 1975) that secondarytype proliferative responses can be generated in vitro. Potential advantages of the PLT method (in which lymphocytes of one individual are “sensitized” in MLC to HLA one haplotype different stimulating cells
This work is supported in part by PHS grant HL-06314 (EJY) and NIH grants AI-00576, AI-08439, CA-16836 and National Foundation-March of Dimes grants CRBS 246 and 6-76-213 (FHB & MLB). This is paper no. 2002 from the Laboratory of Genetics and paper no. 8 7 from the Immunobiology Research Center, The University of Wisconsin, Madison, Wisconsin 53706.
12 NANCY REINSMOEN, EDMOND J . YUNIS, FRITZ H. BACH AND MARILYN L. BACH
and subsequently used as test reagents) are the following. First, PLT cells can be made against essentially any HLA-D haplotype. Second, the test results are available within a day when needed. Third, since it appears that a single D haplotype codes for several determinants (Dupont et al. 1973, Bach et al. 1975), it may be possible to prepare PLT cells against single HLA-D determinants depending on the HLA-D phenotype of responder and stimulator in the primary MLC. It would be of obvious value to have a constant and readily available source of stimulating cells for MLC sensitization that carry a known D locus determinant. Eventually such stimulator cells could be used to sensitize responding cells that lack that determinant but have all other D determinants of the stimulating cell. To that end, we have used lymphoblastoid cell lines from homozygous typing cells as the sensitizing cells in MLC. We have then restimulated the resulting PLT cells with normal peripheral blood lymphocytes from the donor of the lymphoblastoid cell line, other normal lymphocytes carrying the same HLA-D region antigents, as well as cells not carrying those antigens. The results provide preliminary evidence that lymphoblastoid cell lines, which appear to carry the HLA-D determinants (Netzel et al. 1975, Svedmyr et al. 1975), can be used for PLT priming. Materials and methods PLT cells are generated by the method recently described (Sheehy & Bach 1976). In brief, 1 x lo7 responding cells, which are negative for a given DW cluster, were sensitized in separate flasks to either 1 x lo7 normal lymphocytes of the homozygous typing cell donor or 1 x lo6 lymphoblastoid cells from that homozygous typing cell. The primed cells were harvested 10
days later and used for the experiment. PLT cultures were prepared in round bottom wells with 5 x lo4 responding cells and 5 x lo4restimulating cells. The cultures were labeled for 1 6 h amd precipitated (O’Leary et al. 1976). Restimulating cells were used from the original donor of the homozygous typing cell (reference restimulating cells); from cells of the original responding cell donor (control restimulating cells); and from various other individuals (test restimulating cells). Test cells were classified as either carrying the same HLA-D haplotype as the reference cell on the basis of genotyping within a family or, when test cells were used which were unrelated to the control and reference cell, by homozygous typing cell testing.
Results Shown in Figures 1A and 1B are PLT results with two different PLT cells. Results in Figure 1A are obtained using a PLT cell in which cells of a responder that did not carry a given HLA-D cluster were sensitized to the peripheral blood lymphocytes of a homozygous typing cell donor (KH) (Furcht et al. 1976). (This homozygous typing cell does not show a significant association with the presently defined DW clusters.) The PLT cell was restimulated with the normal lymphocytes of the reference cell donor (KH), three family members carrying the same D haplotype (individuals E, F and G), a test cell from an unrelated individual carrying the same D haplotype, by homozygous typing cell testing (individual H), and cells of four individuals (I, J , K and L) not carrying the D cluster by testing with the homozygous cell. The results shown in Figure 1 B are obtained using a PLT cell in which cells from a lymphoblastoid cell line from cells of KH were used for sensitization in the
SENSITIZATION IN PLT 11
13
10
PLT s*nmlmad to
PLT sans811zad L O normal
pmriphmral blood Imuhocyles
lymphoblastoid cell line
n I
: X
t
1
2 Day 01 labding
3 Day of labeling
Figure 1 . Results of PLT studies using PLT cells sensitized t o either normal perpipheral blood leukocytes or the lymphoblastoid cell lines of a homozygous typing cell donor. Cultures were labeled at 24, 4 8 and 72 hours with tritiated thymidine and harvested 16 hours later. The dotted line represents the results obtained with the reference cell, the dashed line the results obtained with test cells carrying the sensitizing DW haplotype and the solid line results obtained with test Celts not carrying the sensitizing DW haplotype. The responding cell donor for experiments shown in 1A and 1B has the HLA phenotype AW23, AW31, B7 in a primary MLC gave a relative response t o KH stimulating cells of 60%. The HLA phenotypes of the eight test restimulating cells are as follows (with the relative response to stimulating cells of KH given in parentheses): E, AW19, BS, BW16 (13); F,AW24, AW31, 8 5 (11); G , AW23, AW31, B5 (13); H, AW31, B5 ( 0 ) ; I, AW19, BW15, BW22 (93); J, A l , A 3 , B 8 , B14, DW3 (69); L, A l , A3, B7, BW15, BW2 (76); K, A3, AW19, B14, B27, DW2 (74). The homozygous typing cell KH is AW24, AW31, BS. The responder for the experiment shown in Figures 1C and I D is AW19, BW1S and BW16 (70) and shows a relative response t o the homozygous stimulating cell, MS, of 76%. The HLA phenotypes of the individuals used in that experiment are as follows (with the relative response t o the homozygous typing cell given in parentheses): M, A l , A3, B7, BW15, DW2 (23); N, A3, AW19, B14, B27, DW2 (21); 0, A2, A3, 8 5 , B7, DW2 (19); P, A2, B7, BW17, DW2 (33);Q, A2, B12, BWlS, DW4 (81); R, A9, A l l , B18, B14 (76); S, A l , A9, B5. BW22 (77); T, A2, AW19, B12, 88 (43). The homozygous typing cell (MS) is A2, A3.87, DW2.
14
NANCY REINSMOEN, EDMOND J. YUNIS, FRITZ H. BACH AND MARILYN L. BACH It
1D
PLT Sensiltzed 10 normal
PLT
1
15
peripheral blood IeukocYIes
sensilired l o Iymphoblasloid c e l l line
-
2
3
Day 01 labeling
primary MLC. Otherwise, the resulting PLT cells were handled identically. The results shown in Figures 1C and 1D are from an experiment in which primary MLC sensitization was accomplished with peripheral blood lymphocytes of a DW2 homozygous typing cell (MS) (Reinsmoen et al. 1975) (Figure 1C) and with the lymphoblastoid cell line of that homozygous typing cell (Figure 1D). Restirnulation of these PLT cells was done with normal peripheral blood lymphocytes of the reference cell donor, four unrelated individuals (M, N , 0 and P) whose cells are positive for DW2 by homozygous typing cell testing) and four unrelated test cells (individuals Q, R , S and T) whose cells are negative for DW2.
Day of Isbeling
Discussion Results obtained with PLT cells appear to measure antigens determined by the HLAD region in that results of PLT tests in unrelated individuals are predictive of primary MLC reactions (Sheehy et al. 1975) and in HLA-B - HLA-D recombinant individuals it is the D region that is important in restimulation (Mawas et al. 1975, Bradley et al. 1976). Whereas one can “type” between 2 x lo3 and 5 x lo3 test cells with PLT cells generated from one pint of blood, it will be of obvious advantage to use a sensitizing cell in the form of a lymphoblastoid cell line for the reasons given in the introduction. Our results, while preliminary, suggest that lymphoblastoid cell lines can be used in
15
SENSITIZATION IN PLT
this way. Lymphoblastoid cell line cells are autostimulatory in the primary MLC (Green & Sell 1970, Knight et al. 1971), however, since we have used normal lymphocytes for the restimulating cell for the PLT test, the autostimulatory effect of the lymphoblastoid cen’ line is probably not pertinent to the results obtained. The normal low level “response” when control restimulating cells are used is consistent with this conclusion. We usually assay response of PLT cells prior to 40 o r 48 h at the latest (Bach et al. 1975, Sheehy et al. 1975, Sheehy & Bach 1976, Sondel et al. 1976), i.e. prior to the appearance of secondary inhibitory effects (as can, for instance, be seen with restimulating cells E and F in Figure 1A). We would, thus, make our comparison of the data obtained in these experiments when the PLT cultures were labeled at 2 4 h for an additional 1 6 h . The possibly greater thymidine incorporation seen in PLT cells prepared against lymphoblastoid cell lines needs further confirmation but may be a useful aspect of this approach. In the experiments shown in Figure 1 as in other experiments we have performed to date, the reference restimulating cell gives maximal or near maximal restimulation, and other test restimulating cells that carry the HLA-D haplotype of the reference cell on the average give higher restimulation that test cells that d o not carry that D haplotype. The findings in these experiments that PLT cells made against lymphoblastoid cell lines give very similar results to those sensitized to normal cells, supports this approach for preparing PLT cells. I t has recently been demonstrated that lymphocytes may be primed against the surface antigens presented o n leukemic cells. A secondary proliferative response can be produced by the leukemic cells which appears to be independent of the
HLA-D determinants of the priming cell (Reinsmoen et al. 1976). These priming and restimulatory antigens are thought to be those that are stimulatory in an MLC (Bach et al. 1969).
Acknowledgements We wish t o thank Drs. Wolfgang Leibold and Richard A. Gatti for establishing the lymphoblastoid cell lines from homozygous typing cells which were from the laboratory of Dr. E.J. Yunis at the University of Minnesota.
References Alter, B.J., GrillotCourvalin. C., Zier, K.S., Sondel, P.M. & Bach, F.H. (1976) Secondary CML: Importance of H-2 LD and S D factors. J . Exp. Med. 143, 1005-1014. Anderson. L.C. & Hayry, P. (1973) Specific priming of mouse thymusdependent lymphocytes to allogeneic cells in vitro. Eur. J. Immunol. 3, 595-599. Bach, M.L.,Bach, F.H. & Joo,P. (1960) Leukemiaassociated antigens in the mixed leukocyte culture test. Science 166, 1520-1522. Bach, F.H., Sondel, P.M.,Sheehy. M.J., Wank, K., Alter, B.J. & Bach, M.L. (1975) The complexity of the HL-A LD system: A PLT analysis, Histocompatibility Testing 1975, ed. Kissmeyer-Nielsen, F., p. 576-580. Munksgaard, Copenhagen. Bradley, B.A., Edwards, J.M., Dunn, D.C. & Calne, R.Y. (1972) Quantitation of mixed lymphocyte reaction by gene dosage phenornenon. Nature New Biology 240, 54-56. Bradley, B.A., Sheehy, M.J., Keuning, J . J . , Termijtelen, A., Franks, D. & van Kood, J.J. (1976) The phenotyping of HLA-D region products by negative and .positive mixed lymphocyte reactions. lmmunogenetics (in press). Dupont, B . , Jersild, C., Hansen, G.S., StaubNielsen, L., Thomsen, M. & Svejgaard, A. (1973) Multiple MLC (LD) determinants o n the same HL-A haplotype. Transplant. Proc. V , 1481-1487. Fradelizi, D. & Dausset, J . (1975) Mixed lympho-
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NANCY REINSMOEN, EDMOND J. YUNIS, FRITZ H. BACH AND MARILYN L. BACH
of several MLR-S (MLC) homozygous cells and the typing for these determinants in the North American Caucasian population (Minnesota). Histocompatibility Testing 1 9 7 5 , ed. Kissmeyer-Nielsen, F., p. 459-463. Munksgaard, Copenhagen. Sheehy, M.J. & Bach, F.H. (1976)Primed LD typing (PLT) - technical considerations. Tissue Antigens (in press). Sheehy, M.J., Sondel, P.M., Bach, M.L., Wank. R. & Bach. F.H. (1975) HL-A LD (lymphocyte defined) typing: A rapid assay with primed 170,989-990. lymphocytes. Science 188,1308-1310. Knight, S.C., Moore, G.E. & Clarkson, B.D. (1971)Stimulation of autochthonous lympho- Sondel, P.M., Sheehy, M.J., Benike, C., Alter, B.J. & Bach. F.H. (1976)Specific detection cytes by cells from normal and leukaemic of HLA-D antigens using primed LD typing lines. Nature New Biol. 229,175-187. (PLT). Transplant. Proc. (in press). MacDonald, H.R., Engers, H.D., Cerottini, J.C. & Brunner, K.T. (1974) Generation of cyto- Svedmyr, E. (1975) Long-term maintenance in vitro of human T cells b y repeated exposure toxic T lymphocytes in vitro. 11. Effect of to the same stimulator cells. Scand. J . repeated exposure to alloantigens on t h e Immunol. 4,421-427. cytotoxic activity of long-term mixed leukocyte cultures. J . Exp. Med. 140. 718-730. Svedmyr. E., Leibold, W. & Gatti, R.A. (1975) Possible use of established cell lines for MLR Mawas, C.E.,Charmot, D., & Sasportes, M. (1975) locus typing. Tissue Antigens 5, 186-195. Secondary response of in vitro primed human lymphocytes to allogeneic cells. 1. Role of Thorsby, E. & Piazza, A. (eds.). (1975) Joint HL-A antigens and mixed lymphocyte reaction report from t h e sixth international histostimulating determinants in secondary in compatibility workshop conference. 11. Typing vitro proliferative response. Immunogenetics for HLA-D (LD-1 or MLC) determinants. 2,449-463. Histocompatibility Testing 1975, ed. KissNetzel, B., Mempel, W.,Albert, E.D., Baumann, P. meyer-Nielsen, F.. p. 414-458. Munksgaard, Copenhagen. & Grosse-Wilde, H. (1975) LD typing in lymphoblastoid cell lines. Immunogenetics 2 , Zier, K.S. & Bach, F.H. (1975) Secondary responses of human lymphocytes t o allo205-210. antigens in vitro. Scand. J. Immunol. 4, O’Leary, J., Reinsmoen, N. & Yunis, E.J. (1976) 607-61 1. Mixed lymphocyte reaction. Manual of Clinical Immunology (in press). Reinsmoen, N., Kersey, J. & Yunis, E.J. (1976) Address: Leukemia-associated antigens detected in N.Heinsmoen primed lymphocyte tests. Transplant. Proc. University of Minnesota (in press). Dept. of Laboratory Medicine and Pathology Reinsmoen, N., Stewart, M., Emme, L., Hanrahan, Medical School L.A., Dupont, B., Hansen, J.A., Friend, P., Box 198,Mayo Memorial Building Amos, D.B. & Yunis, E.J. (1975) Definition Minneapolis, Minnesota 55455
cyte reactivity of human lymphocytes primed in vitro. 1. Secondary response to allogenic lymphocytes. Eur. J . Immunol. 5, 295-301. Furcht, L.T., Chopyk, R.L., Yunis, E.J. & Greenberg, L.J. (1976)Relationship between immune responsiveness in vitro to streptococcal antigens and the HL-1 system in the Chippewa Indians. Fed. Proc 35, 353a. Green, S.S. & Sell, K.W. (1970)Mixed leukocyte stimulation of normal peripheral leukocytes by autologous lymphoblastoid cells. Science
