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Antibodies to non-clustered bovine antigens
Veterinary Immunology and hnmunopathology, 27 ( 1991 ) 91-93
91
Elsevier Science Publishers B.V., Amsterdam
4.10 Antibodies to non-clustered bovine antigens Kathy L. O'Reilly, Linda Eskra and Gary A. Splitter Department of Veterinary Science. Universityof 14"isconsin-Madison, Madison. I,I7 53 706. USA
INTRODUCTION
Many antibodies submitted to the workshop could not be clustered by the definitions established (two antibodies from different laboratories or from different fusions or of different isotypes). Some of these antibodies were to B lymphocytes or monocytes and are discussed elsewhere. This section will deal with certain of those antibodies that were unclustered and reacted with T lymphocytes or non-lineage specific antigens (Table 1 ). Included are mAbs 1-28 to leukocyte common antigen (CD45; Maddox et al., 1985; Mackay et al., 1987), 25-32 to the Pgp-I (CD44; Mackay et al., 1988), 151 (putative CD45), 72-37 (putative CD45R) and 72-87 (putative CDI la). An important aspect of cluster designation in the human is the determination of relative molecular weight (Mr). Some immunoprecipitation data are available for these mAbs. Nine of the eleven mAbs have had the Mr of the antigen determined but only one (8-10C5 ) has had a Mr determined by two laboratories. Other available data included immunofluorescent staining of difi%rent ceil lines and immunohistochemistry. RESULTS
MAbs to lymphocyte sub-populations The results of immunofluorescent staining are given in Appendix 9.3. Biochemical data are summarised in Table 1. Four antibodies, IL-A47, CC28, CC43 and CDC49, stained subpopulations oflymphocytes, i.e. less than 100% of PBM or thymocytes. CC49 and IL-A47 bind small sub-populations of PBM and thymocytes ( < 15% ) while CC43 and CC28 bind much larger populations ( > 30% ). All ofthe IL-A47 ÷ cells express CD2 and either CD4 or CD8 (W.I. Morrison and N.D. MacHugh, unpublished data), indicating that the target antigen is specific for T lymphocytes. Two-colour flow microfluorimetry using anti-T lymphocyte and anti-B lymphocyte antibodies was not performed with the other mAbs. Reactivity on the cell lines and T cell clones shows CC28 and CC49 have simil.'r reactivities and are distinct from CC43 and IL-A47. IL-A47 did not bind Concanavalin A lymphoblqsts (data not 0165-2427/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
92
TABLE
K.L. O'REILLY ET AL.
I
Summary of biochemical results MAb
Isotype
Specificity
Mr
Madison ~
Melbourne 3
ILRAD 2
72-87
G2a
_4
_
IL-A87
G2a
-
-
180, 95 -
possible CDI8
25-32
G 1
-
-
94
CD44
1-28
G2a
-
-
200
CD45
i 51
G2a
-
-
-
CD45
72-37
G I
-
IL-A53
G2a
-
20-96
G 1
-
IL-A47 CC28
G2a G 1
. 150
CC43
G2b
-
CC49
G 1
8-10C5
G2a
110, 140, 2 2 0
possible CDI la
220
possible CD45R
-
-
possible CD45R
85
-
unknown
-
-
12
-
150
-
-
120, 89
85
-
unknown unknown unknown unknown
.
.
or CDI la
.
~Immunoprecipitation from ~25I-labelled bovine lymphoblasts stimulated with Concanavalin 2Immunoprecipitation from ~251-1abelled bovine P B M . 31mmunoprecipitation from ~25I-labelled ovine P B M . 4_, no value available.
A.
shown). None of the antibodies are reactive with the B-lymphoblastoid cell line BL3. Biochemical data are limited (Table l ). The mAbs CC28 and CC49 precipitated a single peptide of 150 kDa while CC43 precipitated a single peptide of 12 kDa.
Pan-leukocyte mAbs The data for this group contains several observations that are difficult to explain. Seven antibodies included in the pan-leukocyte group, 72-87, 1-28, 151, 72-37, 25-32, 8-10C5 and IL-A87, bind to 100% of some bovine or ovine PBM preparation. The first five antibodies were prepared to ovine lymphocytes. It is of interest that 1-28 and 151 do not bind to PBM from all cattle (see Appendix 9.3 ). Both of these antibodies bind to thymocytes and some cell lines ( 1-28; BL-3; 151; B167, B159, B155 and BL-3). Since 1-28 and 151 have been shown to recognise CD45 in sheep, these findings suggest that they react with a polymorphic determinant on the CD45 molecule in cattle. Of interest is the lack of reactivity of these pan-leukocyte antibodies with cell lines and T cell clones. None of these antibodies are reactive with all lines tested. 8-10C5 and 25-32 have nearly identical reactivity patterns. MAb 7237 (putative CD45R) was non-reactive with the ovine cell line and has low reactivity with the T4 and N2 bovine Theileria parva lines but reacted with
ANTIBODIESTONON-CLUSTEREDBOVINEANTIGENS
93
an antigen highly expressed on the BoCD5 + T cell clone T32.6. This mAb and IL-A53, also a putative CD45R, are expressed on B cells (see Naessens and Howard, section 4.8). The most broadly reactive antibody is 72-87 (putative CD1 la) and it reacted weakly with the cell lines B159 (23%), B155 (51%) and T36.7 (45%). IL-A87 is reactive with all lines except B155, B159 and B 169 and stains weakly the undefined clone T36.7. It may detect CD l la or CD 18 as described by Eskra et al. ( 1991 ) and a final conclusion requires further data. Two antibodies, 25-32 and 8-10C5, have a similar distribution except that 25-32 was reported to be absent from bovine granulocytes (Namur). The reported Mr for 8-10C5 are 120, 89 kDa (Madison) and 85 kDa (ILRAD) compared with 94 kDa (Melbourne) for 25-32. Different procedures for immunoprecipitation and different cell types were used making direct comparison difficult. Further data are needed to determine the relationship of these two antibodies. Two other mAbs, GS23A and CH 138A, were submitted as being directed against granulocytes but staining was inconsistent and there was some reactivity with other cells (see Appendix 9.3 ). CONCLUSIONS
These antibodies and others that are non-lineage specific should be included in the next workshop and studied in detail. It is becoming clear that antibodies to the non-lineage specific antigens are very useful in defining functional groups of lymphocytes important in understanding the immune system. Future workshops should include more detailed biochemical analysis (two dimensional PAGE and sequential immunoprecipitations) and functional analysis. The findings in cattle are consistent with 25-32 being to CD44 in cattle as in sheep; 1-28 and 151 reacting with CD45.72-37 may be to a CD45R homologue but further d~ta are needed to be certain. IL-A53 may also be to an epitope on a restricted form of CD45R. MAb 20-96, to CD45R in sheep, appears not to react with a homologous molecule in cattle. MAb 7287 and IL-A87 may react with the bovine CD 1 la and CD 18 homologues but a firm conclusion requires further investigation. REFERENCES Eskra, L., O'Reilly, K.L. and Splitter, G.A., 1991. The bovine p 150/95 molecule (CD 11c/CD 18 ) functions in primary cell-cell interaction. Vet. Immunol. Immunopathol., in press. Maddox, J.F., Mackay, C.R. and Brandon, M.R., 1985. The sheep analogue of leucocyte common antigen. Immunology, 55: 347-353. Mackay, C.R., Maddox, J.F. and Brandon, M.R., 1987. A monoclonal antibody to the p220 component of sheep LCA identifies B cells and a unique lymphocyte subset. Cell. Immunol., 110: 46-55. Mackay, C.R., Maddox, J.F., Wijffel, G.L., Mackay, I.R. and Walker, I.D., 1988. Characterisation of a 95,000 molecule on sheep leukocytes homologous to murine Pgp-l and human CD44. Immunology, 65: 93-99.
91
Elsevier Science Publishers B.V., Amsterdam
4.10 Antibodies to non-clustered bovine antigens Kathy L. O'Reilly, Linda Eskra and Gary A. Splitter Department of Veterinary Science. Universityof 14"isconsin-Madison, Madison. I,I7 53 706. USA
INTRODUCTION
Many antibodies submitted to the workshop could not be clustered by the definitions established (two antibodies from different laboratories or from different fusions or of different isotypes). Some of these antibodies were to B lymphocytes or monocytes and are discussed elsewhere. This section will deal with certain of those antibodies that were unclustered and reacted with T lymphocytes or non-lineage specific antigens (Table 1 ). Included are mAbs 1-28 to leukocyte common antigen (CD45; Maddox et al., 1985; Mackay et al., 1987), 25-32 to the Pgp-I (CD44; Mackay et al., 1988), 151 (putative CD45), 72-37 (putative CD45R) and 72-87 (putative CDI la). An important aspect of cluster designation in the human is the determination of relative molecular weight (Mr). Some immunoprecipitation data are available for these mAbs. Nine of the eleven mAbs have had the Mr of the antigen determined but only one (8-10C5 ) has had a Mr determined by two laboratories. Other available data included immunofluorescent staining of difi%rent ceil lines and immunohistochemistry. RESULTS
MAbs to lymphocyte sub-populations The results of immunofluorescent staining are given in Appendix 9.3. Biochemical data are summarised in Table 1. Four antibodies, IL-A47, CC28, CC43 and CDC49, stained subpopulations oflymphocytes, i.e. less than 100% of PBM or thymocytes. CC49 and IL-A47 bind small sub-populations of PBM and thymocytes ( < 15% ) while CC43 and CC28 bind much larger populations ( > 30% ). All ofthe IL-A47 ÷ cells express CD2 and either CD4 or CD8 (W.I. Morrison and N.D. MacHugh, unpublished data), indicating that the target antigen is specific for T lymphocytes. Two-colour flow microfluorimetry using anti-T lymphocyte and anti-B lymphocyte antibodies was not performed with the other mAbs. Reactivity on the cell lines and T cell clones shows CC28 and CC49 have simil.'r reactivities and are distinct from CC43 and IL-A47. IL-A47 did not bind Concanavalin A lymphoblqsts (data not 0165-2427/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
92
TABLE
K.L. O'REILLY ET AL.
I
Summary of biochemical results MAb
Isotype
Specificity
Mr
Madison ~
Melbourne 3
ILRAD 2
72-87
G2a
_4
_
IL-A87
G2a
-
-
180, 95 -
possible CDI8
25-32
G 1
-
-
94
CD44
1-28
G2a
-
-
200
CD45
i 51
G2a
-
-
-
CD45
72-37
G I
-
IL-A53
G2a
-
20-96
G 1
-
IL-A47 CC28
G2a G 1
. 150
CC43
G2b
-
CC49
G 1
8-10C5
G2a
110, 140, 2 2 0
possible CDI la
220
possible CD45R
-
-
possible CD45R
85
-
unknown
-
-
12
-
150
-
-
120, 89
85
-
unknown unknown unknown unknown
.
.
or CDI la
.
~Immunoprecipitation from ~25I-labelled bovine lymphoblasts stimulated with Concanavalin 2Immunoprecipitation from ~251-1abelled bovine P B M . 31mmunoprecipitation from ~25I-labelled ovine P B M . 4_, no value available.
A.
shown). None of the antibodies are reactive with the B-lymphoblastoid cell line BL3. Biochemical data are limited (Table l ). The mAbs CC28 and CC49 precipitated a single peptide of 150 kDa while CC43 precipitated a single peptide of 12 kDa.
Pan-leukocyte mAbs The data for this group contains several observations that are difficult to explain. Seven antibodies included in the pan-leukocyte group, 72-87, 1-28, 151, 72-37, 25-32, 8-10C5 and IL-A87, bind to 100% of some bovine or ovine PBM preparation. The first five antibodies were prepared to ovine lymphocytes. It is of interest that 1-28 and 151 do not bind to PBM from all cattle (see Appendix 9.3 ). Both of these antibodies bind to thymocytes and some cell lines ( 1-28; BL-3; 151; B167, B159, B155 and BL-3). Since 1-28 and 151 have been shown to recognise CD45 in sheep, these findings suggest that they react with a polymorphic determinant on the CD45 molecule in cattle. Of interest is the lack of reactivity of these pan-leukocyte antibodies with cell lines and T cell clones. None of these antibodies are reactive with all lines tested. 8-10C5 and 25-32 have nearly identical reactivity patterns. MAb 7237 (putative CD45R) was non-reactive with the ovine cell line and has low reactivity with the T4 and N2 bovine Theileria parva lines but reacted with
ANTIBODIESTONON-CLUSTEREDBOVINEANTIGENS
93
an antigen highly expressed on the BoCD5 + T cell clone T32.6. This mAb and IL-A53, also a putative CD45R, are expressed on B cells (see Naessens and Howard, section 4.8). The most broadly reactive antibody is 72-87 (putative CD1 la) and it reacted weakly with the cell lines B159 (23%), B155 (51%) and T36.7 (45%). IL-A87 is reactive with all lines except B155, B159 and B 169 and stains weakly the undefined clone T36.7. It may detect CD l la or CD 18 as described by Eskra et al. ( 1991 ) and a final conclusion requires further data. Two antibodies, 25-32 and 8-10C5, have a similar distribution except that 25-32 was reported to be absent from bovine granulocytes (Namur). The reported Mr for 8-10C5 are 120, 89 kDa (Madison) and 85 kDa (ILRAD) compared with 94 kDa (Melbourne) for 25-32. Different procedures for immunoprecipitation and different cell types were used making direct comparison difficult. Further data are needed to determine the relationship of these two antibodies. Two other mAbs, GS23A and CH 138A, were submitted as being directed against granulocytes but staining was inconsistent and there was some reactivity with other cells (see Appendix 9.3 ). CONCLUSIONS
These antibodies and others that are non-lineage specific should be included in the next workshop and studied in detail. It is becoming clear that antibodies to the non-lineage specific antigens are very useful in defining functional groups of lymphocytes important in understanding the immune system. Future workshops should include more detailed biochemical analysis (two dimensional PAGE and sequential immunoprecipitations) and functional analysis. The findings in cattle are consistent with 25-32 being to CD44 in cattle as in sheep; 1-28 and 151 reacting with CD45.72-37 may be to a CD45R homologue but further d~ta are needed to be certain. IL-A53 may also be to an epitope on a restricted form of CD45R. MAb 20-96, to CD45R in sheep, appears not to react with a homologous molecule in cattle. MAb 7287 and IL-A87 may react with the bovine CD 1 la and CD 18 homologues but a firm conclusion requires further investigation. REFERENCES Eskra, L., O'Reilly, K.L. and Splitter, G.A., 1991. The bovine p 150/95 molecule (CD 11c/CD 18 ) functions in primary cell-cell interaction. Vet. Immunol. Immunopathol., in press. Maddox, J.F., Mackay, C.R. and Brandon, M.R., 1985. The sheep analogue of leucocyte common antigen. Immunology, 55: 347-353. Mackay, C.R., Maddox, J.F. and Brandon, M.R., 1987. A monoclonal antibody to the p220 component of sheep LCA identifies B cells and a unique lymphocyte subset. Cell. Immunol., 110: 46-55. Mackay, C.R., Maddox, J.F., Wijffel, G.L., Mackay, I.R. and Walker, I.D., 1988. Characterisation of a 95,000 molecule on sheep leukocytes homologous to murine Pgp-l and human CD44. Immunology, 65: 93-99.