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Analyses of monoclonal antibodies reacting with porcine wCD6: Results from the Second International Swine CD workshop
Veterinary Immunology and Immunopathology 60 Ž1998. 285–289
Analyses of monoclonal antibodies reacting with porcine wCD6: Results from the Second International Swine CD workshop M.D. Pescovitz a,) , B.K. Book a , B. Aasted b, J. Dominguez c , R. Bullido c , I. Trebichavsky d , B. Novikov e, I. Valpotic f , J. Nielsen g , S. Arn h , D.H. Sachs h , J.K. Lunney i , P.C. Boyd i , l J. Walker j, R. Lee j, N. Petrinec k , A. Saalmuller ¨ a
h
Indiana UniÕersity, Dept. of Surgery and Microbiologyr Immunology, Indianapolis, IN 46202, USA b Royal Veterinary and Agricultural UniÕersity, Frederiksberg C, Denmark c Centro de InÕestigaciones en Sanidad Animal, INIA, Valdeolmos, Madrid, Spain d Institute Microbiology, Prague, Czech Republic e All-Russian Research Institute of Veterinary Virology and Microbiology, Vlasimizskaya obl., Russian Federation f Department of Biology, Veterinary Faculty, UniÕersity of Zagreb, Zagreb, Croatia g Danish Veterinary Institute for Virus Research, Lindholm, KalÕehaÕe, Denmark Transplantation Biology Research Center, Massachusetts General Hospital, Charlestown, MA, USA i Immunology and Disease Resistance Laboratory, LPSI, ARS, USDA, BeltsÕille, MD, USA j Center for Animal Biotechnology, UniÕersity of Melbourne, Victoria, Australia k Institute of Immunology, Zagreb, Croatia l Federal Research Center for Virus Diseases of Animals, Tubingen, Germany ¨
Abstract Among the 57 monoclonal antibodies analyzed within the T-cell group of the Second International Swine CD Workshop, one mAb fell within cluster T14a that included the CD6 standard a38b2 ŽNo. 175.. The new mAb MIL8 ŽNo. 082. and a38b2 both precipitated from activated T-cells a 150 kDa monomeric protein. Staining patterns on the various cell types were similar. There was no inhibition of binding of either mAb to peripheral blood T-cells with the opposite mAb. The new mAb, MIL8, reacts with a separate epitope on porcine wCD6. q 1998 Elsevier Science B.V. Keywords: Monoclonal antibodies; T-cells; porcine wCD6
)
Corrresponding author.
0165-2427r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 2 4 2 7 Ž 9 7 . 0 0 1 0 5 - 0
286
M.D. PescoÕitz et al.r Veterinary Immunology and Immunopathology 60 (1998) 285–289
1. Introduction The CD6 molecule is reported to be a 130 kDa protein present on all human T-cells, most thymocytes, and some B-cells ŽReinherz et al., 1979; Morimoto et al., 1988.. The protein itself has a mw of 88 kDa with the additional weight from added N-linked glycan and O-linked oligosaccharides ŽSwack et al., 1991.. CD6 has sequence homology with CD5 and with the mouse type I macrophage scavenger receptor ŽAruffo et al., 1991.. Recently the Xenopus CD5 protein has also shown to be homologous to this receptor ŽJurgens et al., 1995.. CD6 in humans has been shown to provide an activation signal to suboptimally stimulated cells. In the First Swine CD Workshop, 2 mAb were identified, which based on their reactivity with cell populations, and on the molecular weight of the precipitated antigen for one Ža38b2, No. 175., were assigned to wCD6a ŽSaalmuller et al., 1994.. ¨ As detailed in this journal, the Second International Porcine CD Workshop was held in Davis, CA. Of the 165 new and 19 standard mAb submitted, 57 mAb including 11 mAb standards were determined to be reactive with T-cells. One of these, No. 082 ŽMIL8. grouped closely with the internal anti-wCD6 standard No. 175 Ža38b2. ŽTable 1.. Further analysis, including the determination of the molecular weight of the target antigen and epitope blocking studies, confirmed that the new mAb reacts with the same protein but a different epitope, as the anti-wCD6a mAb, a38b2. 2. Methods One color flow cytometric analysis was performed with the two anti-CD6 mAb and anti-mouse FITC. Inhibition studies were performed using peripheral blood T-cells as the target. Briefly, cells were incubated first with one of the unlabelled test mAb, followed by incubation with the other unlabelled mAb. The cells were then probed with isotype specific FITC-conjugated antiserum Žanti-IgG2a for MIL8, and anti-IgG1 for a38b2.. Immunoprecipitation using the standard anti-wCD6, a38b2, and the new mAb MIL8 with lysates of Con-A blasts was performed as described elsewhere in this volume ŽAasted et al., 1998.. 3. Results and discussion The one color flow cytometry patterns on peripheral blood lymphocytes shows similar staining of the wCD6 standard and MIL8 ŽFig. 1a.. The inhibition results ŽFig. 1a. indicated that neither mAb inhibited binding of the other to peripheral T-cells,
Table 1 Summary of monoclonal antibodies within cluster T14a Workshop number
mAb
Donor laboratory
Ig isotype
Molecular mass
082 175
MIL8 a38b2
Stokes Saalmuller ¨
IgG2a IgG1
150 150
Molecular weights were determined by immunoprecipitation from activated T-cells.
M.D. PescoÕitz et al.r Veterinary Immunology and Immunopathology 60 (1998) 285–289
Fig. 1. Characterization of wCD6 epitopes. Ža. Resting T-cells were stained with either a38b2 and an IgG1-specific PE-conjugated antiserum Žtop row. or with MIL8 and an IgG2a-specific FITC conjugated antiserum Žbottom row.. The first column demonstrated fluorescence intensity without addition of the alternate mAb whereas the second column demonstrates fluorescence with the alternate mAb. Grey colored curves represent background staining without primary antibody. There was no inhibition of staining detectable. Žb. Coexpression of MIL8 and a38b2 epitopes on porcine T-cells. Staining was as in Ža. above except that all reagents were added for this analysis. There is complete concordance of expression of the two epitopes.
287
288
M.D. PescoÕitz et al.r Veterinary Immunology and Immunopathology 60 (1998) 285–289
whereas each mAb completely inhibited its own binding Ždata not shown.. When cells were probed with both anti-CD6 mAb and counter stained with the isotype specific reagents, the cells were found to lie on the diagonal thus indicating that the mAb apparently recognized different epitopes of the same molecule ŽFig. 1b.. Of concern however with the wCD6 designation is the mw of the precipitated antigen. Although both mAb precipitated the same mw protein, thereby further confirming the reactivity with the same cell surface protein, the mw was 150 kDa as compared to the previous report of 110 kDa for porcine CD6 ŽSaalmuller et al., 1994; Pauly et al., 1996.. Since ¨ the core protein of human CD6 has a mw of only 88 kDa ŽSwack et al., 1991., it is possible that the differences in mw between pig and human CD6 and between the two different reports of pig CD6 may result from glycosylation differences based on the population. In fact the mw determined with the current Workshop used Con A blasts as the cell from which CD6 was immunoprecipitated while the previous report used peripheral resting T-cells. In preliminary results Ždata not shown. the mw of the protein precipitated with the two wCD6 mAbs was lower Ž100–115 kDa. in resting cells than simultaneously run Con A blasts. Furthermore, there were multiple bands possibly reflecting glycosylation versus phosphorylation differences. Although not part of the current workshop, wCD6 has been found to be differentially expressed on porcine peripheral T-cell populations ŽSaalmuller et al., 1994.. All of the ¨ wCD4q cells were found to be in the wCD6q population while the wCD8q cells were split between the wCD6y and wCD6q populations. This differential expression perhaps explains why the wCD6 mAb so tightly clustered with the wCD4 mAb ŽPescovitz et al., 1998.. This further emphasizes the importance of collecting additional information, such as immunoprecipitation of the target antigen, when assigning CD antigen reactivity. Because of the data from this workshop, MIL8 ŽNo. 082. has been assigned to the same cluster as a38b2, but based on the epitope analysis, MIL8 defines a new epitope of porcine wCD6. Because of the lack of sequence data of the target antigen, and given the molecular weight that differs from the reported human CD6, a38b2 and MIL8 are assigned as reactive with wCD6. References Aasted, B., Gori, K., Dominguez, J., Ezquerra, A., Bullido, R., Arn, S., Bianchi, A., Binns, R., Chu, R.M., Davis, W.C., Denham, S., Haverson, K., Jensen, K.T., Kim, Y.B., Magyar, A., Petersen, K.R., Saalmuller, ¨ A., Sachs, D., Schutt, ¨ C., Shimizu, M., Stokes, C., Whittall, T., Yang, H., Zuckermann, F., 1998. Immunoprecipitation studies of monoclonal antibodies submitted to the Second International Swine CD Workshop. Vet. Immunol. Immunopathol. 60, 229–235. Aruffo, A., Melnick, M.B., Linsley, P.S., Seed, B., 1991. The lymphocyte glycoprotein CD6 contains a repeated domain structure characteristic of a new family of cell surface and secreted proteins. J. Exp. Med. 174, 949–952. Jurgens, J.B., Gartland, L.A., Du Pasquier, L., Horton, J.D., Goebel, T.W.F., Cooper, M.D., 1995. Identification of a candidate CD5 homologue in the amphibian Xenopus laeÕie. J. Immunol. 155, 4218–4223. Morimoto, C., Rudd, C.E., Letvin, N.L., Hagan, M., Schlossman, S.F., 1988. 2HI—a novel antigen involved in T lymphocyte triggering. J. Immunol. 140, 2165–2170. Pauly, T., Weiland, E., Hirt, W., Dreyer-Bux, C., Maurer, S., Summerfield, A., Saalmueller, A., 1996. Differentiation between MHC-restricted and non-MHC-restricted porcine cytolytic T lymphocytes. Immunology 88, 238–246.
M.D. PescoÕitz et al.r Veterinary Immunology and Immunopathology 60 (1998) 285–289
289
Pescovitz, M.D., Book, B.K., Aasted, B., Dominguez, J., Ezquerra, A., Trebichavsky, I., Novikov, B., Valpotic, I., Sver, L., Nielsen, J., Arn, S., Sachs, D.H., Lunneyk, J.K., Boyd, P.C., Walker, J., Lee, R., Davis, W.C., Barbosa, I.R., Zuckermann, F., Saalmuller, A., 1998. Summary of workshop findings for ¨ antibodies reacting with porcine T-cells and activation antigens: results from the Second International Swine CD Workshop. Vet. Immunol. Immunopathol. 60, 249–257. Reinherz, E.L., Kung, P.C., Goldstein, G., Schlossman, S.F., 1979. A monoclonal antibody with selective reactivity with functionally mature human thymocytes and all peripheral human T cells. J. Immunol. 123, 1312–1317. Saalmuller, A., Aasted, B., Canals, A., Dominguez, J., Goldman, T., Lunney, J.K., Maurer, S., Pauly, T., ¨ Pescovitz, M.D., Pospisil, R., Salmon, H., Trebichavsky, I., Valpotic, I., Vizcaino, J.S., Weiland, E., Zuckermann, F., 1994. Analyses of monoclonal antibodies reactive with porcine CD6. Vet. Immunol. Immunopathol. 43, 243–247. Swack, J.A., Mier, J.W., Romain, P.L., Hull, S.R., Rudd, C.E., 1991. Biosynthesis and post-translational modification of CD6, a T cell signal-transducing molecule. J. Biol. Chem. 266, 7137–7143.
Analyses of monoclonal antibodies reacting with porcine wCD6: Results from the Second International Swine CD workshop M.D. Pescovitz a,) , B.K. Book a , B. Aasted b, J. Dominguez c , R. Bullido c , I. Trebichavsky d , B. Novikov e, I. Valpotic f , J. Nielsen g , S. Arn h , D.H. Sachs h , J.K. Lunney i , P.C. Boyd i , l J. Walker j, R. Lee j, N. Petrinec k , A. Saalmuller ¨ a
h
Indiana UniÕersity, Dept. of Surgery and Microbiologyr Immunology, Indianapolis, IN 46202, USA b Royal Veterinary and Agricultural UniÕersity, Frederiksberg C, Denmark c Centro de InÕestigaciones en Sanidad Animal, INIA, Valdeolmos, Madrid, Spain d Institute Microbiology, Prague, Czech Republic e All-Russian Research Institute of Veterinary Virology and Microbiology, Vlasimizskaya obl., Russian Federation f Department of Biology, Veterinary Faculty, UniÕersity of Zagreb, Zagreb, Croatia g Danish Veterinary Institute for Virus Research, Lindholm, KalÕehaÕe, Denmark Transplantation Biology Research Center, Massachusetts General Hospital, Charlestown, MA, USA i Immunology and Disease Resistance Laboratory, LPSI, ARS, USDA, BeltsÕille, MD, USA j Center for Animal Biotechnology, UniÕersity of Melbourne, Victoria, Australia k Institute of Immunology, Zagreb, Croatia l Federal Research Center for Virus Diseases of Animals, Tubingen, Germany ¨
Abstract Among the 57 monoclonal antibodies analyzed within the T-cell group of the Second International Swine CD Workshop, one mAb fell within cluster T14a that included the CD6 standard a38b2 ŽNo. 175.. The new mAb MIL8 ŽNo. 082. and a38b2 both precipitated from activated T-cells a 150 kDa monomeric protein. Staining patterns on the various cell types were similar. There was no inhibition of binding of either mAb to peripheral blood T-cells with the opposite mAb. The new mAb, MIL8, reacts with a separate epitope on porcine wCD6. q 1998 Elsevier Science B.V. Keywords: Monoclonal antibodies; T-cells; porcine wCD6
)
Corrresponding author.
0165-2427r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 2 4 2 7 Ž 9 7 . 0 0 1 0 5 - 0
286
M.D. PescoÕitz et al.r Veterinary Immunology and Immunopathology 60 (1998) 285–289
1. Introduction The CD6 molecule is reported to be a 130 kDa protein present on all human T-cells, most thymocytes, and some B-cells ŽReinherz et al., 1979; Morimoto et al., 1988.. The protein itself has a mw of 88 kDa with the additional weight from added N-linked glycan and O-linked oligosaccharides ŽSwack et al., 1991.. CD6 has sequence homology with CD5 and with the mouse type I macrophage scavenger receptor ŽAruffo et al., 1991.. Recently the Xenopus CD5 protein has also shown to be homologous to this receptor ŽJurgens et al., 1995.. CD6 in humans has been shown to provide an activation signal to suboptimally stimulated cells. In the First Swine CD Workshop, 2 mAb were identified, which based on their reactivity with cell populations, and on the molecular weight of the precipitated antigen for one Ža38b2, No. 175., were assigned to wCD6a ŽSaalmuller et al., 1994.. ¨ As detailed in this journal, the Second International Porcine CD Workshop was held in Davis, CA. Of the 165 new and 19 standard mAb submitted, 57 mAb including 11 mAb standards were determined to be reactive with T-cells. One of these, No. 082 ŽMIL8. grouped closely with the internal anti-wCD6 standard No. 175 Ža38b2. ŽTable 1.. Further analysis, including the determination of the molecular weight of the target antigen and epitope blocking studies, confirmed that the new mAb reacts with the same protein but a different epitope, as the anti-wCD6a mAb, a38b2. 2. Methods One color flow cytometric analysis was performed with the two anti-CD6 mAb and anti-mouse FITC. Inhibition studies were performed using peripheral blood T-cells as the target. Briefly, cells were incubated first with one of the unlabelled test mAb, followed by incubation with the other unlabelled mAb. The cells were then probed with isotype specific FITC-conjugated antiserum Žanti-IgG2a for MIL8, and anti-IgG1 for a38b2.. Immunoprecipitation using the standard anti-wCD6, a38b2, and the new mAb MIL8 with lysates of Con-A blasts was performed as described elsewhere in this volume ŽAasted et al., 1998.. 3. Results and discussion The one color flow cytometry patterns on peripheral blood lymphocytes shows similar staining of the wCD6 standard and MIL8 ŽFig. 1a.. The inhibition results ŽFig. 1a. indicated that neither mAb inhibited binding of the other to peripheral T-cells,
Table 1 Summary of monoclonal antibodies within cluster T14a Workshop number
mAb
Donor laboratory
Ig isotype
Molecular mass
082 175
MIL8 a38b2
Stokes Saalmuller ¨
IgG2a IgG1
150 150
Molecular weights were determined by immunoprecipitation from activated T-cells.
M.D. PescoÕitz et al.r Veterinary Immunology and Immunopathology 60 (1998) 285–289
Fig. 1. Characterization of wCD6 epitopes. Ža. Resting T-cells were stained with either a38b2 and an IgG1-specific PE-conjugated antiserum Žtop row. or with MIL8 and an IgG2a-specific FITC conjugated antiserum Žbottom row.. The first column demonstrated fluorescence intensity without addition of the alternate mAb whereas the second column demonstrates fluorescence with the alternate mAb. Grey colored curves represent background staining without primary antibody. There was no inhibition of staining detectable. Žb. Coexpression of MIL8 and a38b2 epitopes on porcine T-cells. Staining was as in Ža. above except that all reagents were added for this analysis. There is complete concordance of expression of the two epitopes.
287
288
M.D. PescoÕitz et al.r Veterinary Immunology and Immunopathology 60 (1998) 285–289
whereas each mAb completely inhibited its own binding Ždata not shown.. When cells were probed with both anti-CD6 mAb and counter stained with the isotype specific reagents, the cells were found to lie on the diagonal thus indicating that the mAb apparently recognized different epitopes of the same molecule ŽFig. 1b.. Of concern however with the wCD6 designation is the mw of the precipitated antigen. Although both mAb precipitated the same mw protein, thereby further confirming the reactivity with the same cell surface protein, the mw was 150 kDa as compared to the previous report of 110 kDa for porcine CD6 ŽSaalmuller et al., 1994; Pauly et al., 1996.. Since ¨ the core protein of human CD6 has a mw of only 88 kDa ŽSwack et al., 1991., it is possible that the differences in mw between pig and human CD6 and between the two different reports of pig CD6 may result from glycosylation differences based on the population. In fact the mw determined with the current Workshop used Con A blasts as the cell from which CD6 was immunoprecipitated while the previous report used peripheral resting T-cells. In preliminary results Ždata not shown. the mw of the protein precipitated with the two wCD6 mAbs was lower Ž100–115 kDa. in resting cells than simultaneously run Con A blasts. Furthermore, there were multiple bands possibly reflecting glycosylation versus phosphorylation differences. Although not part of the current workshop, wCD6 has been found to be differentially expressed on porcine peripheral T-cell populations ŽSaalmuller et al., 1994.. All of the ¨ wCD4q cells were found to be in the wCD6q population while the wCD8q cells were split between the wCD6y and wCD6q populations. This differential expression perhaps explains why the wCD6 mAb so tightly clustered with the wCD4 mAb ŽPescovitz et al., 1998.. This further emphasizes the importance of collecting additional information, such as immunoprecipitation of the target antigen, when assigning CD antigen reactivity. Because of the data from this workshop, MIL8 ŽNo. 082. has been assigned to the same cluster as a38b2, but based on the epitope analysis, MIL8 defines a new epitope of porcine wCD6. Because of the lack of sequence data of the target antigen, and given the molecular weight that differs from the reported human CD6, a38b2 and MIL8 are assigned as reactive with wCD6. References Aasted, B., Gori, K., Dominguez, J., Ezquerra, A., Bullido, R., Arn, S., Bianchi, A., Binns, R., Chu, R.M., Davis, W.C., Denham, S., Haverson, K., Jensen, K.T., Kim, Y.B., Magyar, A., Petersen, K.R., Saalmuller, ¨ A., Sachs, D., Schutt, ¨ C., Shimizu, M., Stokes, C., Whittall, T., Yang, H., Zuckermann, F., 1998. Immunoprecipitation studies of monoclonal antibodies submitted to the Second International Swine CD Workshop. Vet. Immunol. Immunopathol. 60, 229–235. Aruffo, A., Melnick, M.B., Linsley, P.S., Seed, B., 1991. The lymphocyte glycoprotein CD6 contains a repeated domain structure characteristic of a new family of cell surface and secreted proteins. J. Exp. Med. 174, 949–952. Jurgens, J.B., Gartland, L.A., Du Pasquier, L., Horton, J.D., Goebel, T.W.F., Cooper, M.D., 1995. Identification of a candidate CD5 homologue in the amphibian Xenopus laeÕie. J. Immunol. 155, 4218–4223. Morimoto, C., Rudd, C.E., Letvin, N.L., Hagan, M., Schlossman, S.F., 1988. 2HI—a novel antigen involved in T lymphocyte triggering. J. Immunol. 140, 2165–2170. Pauly, T., Weiland, E., Hirt, W., Dreyer-Bux, C., Maurer, S., Summerfield, A., Saalmueller, A., 1996. Differentiation between MHC-restricted and non-MHC-restricted porcine cytolytic T lymphocytes. Immunology 88, 238–246.
M.D. PescoÕitz et al.r Veterinary Immunology and Immunopathology 60 (1998) 285–289
289
Pescovitz, M.D., Book, B.K., Aasted, B., Dominguez, J., Ezquerra, A., Trebichavsky, I., Novikov, B., Valpotic, I., Sver, L., Nielsen, J., Arn, S., Sachs, D.H., Lunneyk, J.K., Boyd, P.C., Walker, J., Lee, R., Davis, W.C., Barbosa, I.R., Zuckermann, F., Saalmuller, A., 1998. Summary of workshop findings for ¨ antibodies reacting with porcine T-cells and activation antigens: results from the Second International Swine CD Workshop. Vet. Immunol. Immunopathol. 60, 249–257. Reinherz, E.L., Kung, P.C., Goldstein, G., Schlossman, S.F., 1979. A monoclonal antibody with selective reactivity with functionally mature human thymocytes and all peripheral human T cells. J. Immunol. 123, 1312–1317. Saalmuller, A., Aasted, B., Canals, A., Dominguez, J., Goldman, T., Lunney, J.K., Maurer, S., Pauly, T., ¨ Pescovitz, M.D., Pospisil, R., Salmon, H., Trebichavsky, I., Valpotic, I., Vizcaino, J.S., Weiland, E., Zuckermann, F., 1994. Analyses of monoclonal antibodies reactive with porcine CD6. Vet. Immunol. Immunopathol. 43, 243–247. Swack, J.A., Mier, J.W., Romain, P.L., Hull, S.R., Rudd, C.E., 1991. Biosynthesis and post-translational modification of CD6, a T cell signal-transducing molecule. J. Biol. Chem. 266, 7137–7143.