- Email: [email protected]
Summary of workshop findings for antibodies reacting with porcine T-cells and activation antigens: results from the Second International Swine CD Workshop
Veterinary Immunology and Immunopathology 60 Ž1998. 251–260
Summary of workshop findings for antibodies reacting with porcine T-cells and activation antigens: results from the Second International Swine CD Workshop M.D. Pescovitz a,) , B.K. Book a , B. Aasted b, J. Dominguez c , A. Ezquerra c , I. Trebichavsky d , B. Novikov e, I. Valpotic f , L. Sver f , J. Nielsen g , S. Arn h , D.H. Sachs h , J.K. Lunney i , P.C. Boyd i , J. Walker j, R. Lee j, William Davis k , I.R. Barbosa k , m F. Zuckermann l , A. Saalmuller ¨ a
Indiana UniÕersity, Department 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 h 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 Washington State UniÕersity, Pullman, WA, USA l Department of Veterinary Pathology, UniÕersity of Illinois, Urbana, IL, USA m Federal Research Center for Virus Diseases of Animals, Tubingen, Germany ¨
Abstract After initial evaluation of the 176 new and 19 control monoclonal antibodies ŽmAb. submitted to the Second International Swine CD Workshop, 57 were assigned to the T-cellractivation marker subgroup. These 57 mAb were further analyzed using flow cytometry on whole blood lymphocytes, splenocytes, Peyer’s patch lymphocytes, in vitro cell lines, broncho-alveolar lavage cells, Con A and PHA blasts, fetal cell populations, and by 2-color flow cytometry against mAb to
)
Corresponding 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 1 - 3
252
M.D. PescoÕitz et al.r Veterinary Immunology and Immunopathology 60 (1998) 251–260
porcine CD2, CD4, and CD8. Finally, the molecular weights of the target antigens were characterized when possible. As a result of these analyses, 23 mAb were distributed into 7 CD clusters. Newly confirmed mAb assignments included: two CD2; one CD4; two CD5; one wCD6; and one wCD25. Three new mAb were found that reacted with wCD8, one of which defined a new epitope, wCD8c. For the first time, mAb against porcine CD3 were identified, including 6 mAb that reacted with three different epitopes. Several new mAb reacted with antigens whose expression varied depending on the activation state of the test cell. These will require further characterization in order to assign a CD number. q 1998 Elsevier Science B.V. Keywords: Monoclonal antibodies; T-cells; Swine CD workshop; Porcine
1. Introduction International workshops designed to categorize into Clusters of Differentiation ŽCD. large numbers of independently generated monoclonal antibodies against lymphocyte populations, have been extremely successful. Following this pattern, the First International Swine CD Workshop confirmed the assignment of mAb available at that time to well-defined CD groups, some of which were analogous to those seen with other species, and some unique to the pig ŽCarr et al., 1994. ŽSaalmuller et al., 1994.. The ¨ success of this first workshop, and the recognized need to identify other mAb to CD described in other species, prompted the organization of the Second International Swine CD Workshop. This workshop, orchestrated by Dr. Armin Saalmuller, analyzed 176 ¨ mAb in a first round of analyses. These analyses assigned 51 of the 176 mAb to 6 clusters, which appeared to react with T-cells or activation markers ŽSaalmuller et al., ¨ 1998.. Six additional mAb, which were not assigned to these clusters but according the opinion of the submitting laboratory were T-cell reactive, were included in the subsequent analyses. These additional analyses confirmed the assignment of most of the mAb as reactive with T-cell or activation antigens and further grouped the mAb into 7 well-defined CD groups.
2. Methods Fifty-seven mAb identified as reacting with T-cells or activation markers, including 11 standards ŽSaalmuller ¨ et al., 1998., were distributed to the authors of this paper. The mAb were then tested by one, and in some cases 2 color, flow cytometry. The cell populations used for one color immunostaining included both post natal and fetal thymocytes, splenocytes, lymphocytes from mesenteric lymph nodes and Peyer’s Patches, peripheral blood mononuclear cells, white blood cells, and bone marrow cells. Various in vitro porcine cell lines and both PHA and Con A blasts were also studied. Two color Fig. 1. Dendogram of mAb analyzed by the T-cellrActivation group. The clusters that have been assigned are labeled T1 to T14b. All mAb that did not cluster with at least one other mAb are labeled ‘U’. The small numbers to the left are the assigned workshop numbers; the corresponding mAb designations from the submitting laboratories are listed in Table 1.
M.D. PescoÕitz et al.r Veterinary Immunology and Immunopathology 60 (1998) 251–260
253
254
M.D. PescoÕitz et al.r Veterinary Immunology and Immunopathology 60 (1998) 251–260
analyses were performed with the workshop mAb, counter staining with anti-pig Ig to exclude B-cells from the analysis, and against standard anti-CD2, CD4, and CD8 mAb. These analyses gave 39 data sets, which were further analyzed by the Database IV software from the Fourth Human Leukocyte Workshop. The clustering after this analysis is shown in Fig. 1. Further definition of the clusters was aided by the determination of the molecular weight of the target cell antigen ŽAasted et al., 1998.. For mAbs assigned to the clusters T12 containing the CD2 standard, and cluster T14a and T14b containing the CD4 and wCD6 standards epitope analysis was performed. Peripheral blood lymphocytes were first incubated with the workshop antibody to be tested or appropriate control antibodies. Then without washing, suboptimal amounts of either biotin conjugated anti-wCD2 or anti-wCD4 mAb were added. The cells were then washed and stained with streptavidin-PE. 3. Results The reactivity of the 57 mAb with representative cell populations is summarized in Table 1. Thirty-five mAb were grouped into 14 well-defined clusters of at least 2 mAb ŽFig. 1.. Five mAb ŽPGBL31A, No. 044; IAH-CC57, No. 125; UCP1C10, No. 131; C28, No. 145; and FQ3E8, No. 158. clustered tightly in T4 but their staining was very weak at best and the clustering may have been an artifact. Seventeen mAb Žnoted as ‘U’ in Fig. 1. did not cluster with any other mAb. These included several of the internal standards: 76-7-4 ŽNo. 171, CD1.; 11r8r1 ŽNo. 183, SWC1.; and MAC319 ŽNo. 186, SWC4.. Two mAb BB6-10A10 ŽNo. 168. and K139.3E1 ŽNo. 085. were further analyzed in the B-cell section. MAb PGBL2A ŽNo. 037. appeared to block binding of the standard anti-CD2 mAb, MSA4, but it did not cluster in T12 with the CD2 standard, and the molecular weight of the target antigen was not known. However, it is thought that this mAb is anti-Con A ŽDavis, unpublished results.. mAb PGBL13A ŽNo. 047. and PG104A ŽNo. 024. immunoprecipitated a similar molecular weight protein of 14r16 kDa ŽAasted et al., 1998. but clearly gave different reactivity patterns with intact cells. 4. Porcine CD2 Three mAb distributed to cluster T12 including the standard anti-CD2 mAb MSA4 ŽNo. 172.. The two new mAb PGBL6A ŽNo. 046. and PGBL23A ŽNo. 048. inhibited binding of biotinylated MSA4 but not anti-CD4 ŽTable 2.. In neither case was the target antigen immunoprecipitated. On the basis of on the clustering and the inhibition, however, these mAb have been assigned to CD2a. 4.1. Porcine CD3 Six monoclonal antibodies distributed into cluster T10 and T11. There were no previously identified standards with which to compare these mAb. All of the mAb precipitated a 24 kDa protein on reduced polyacrylamide gels. mAbs FY2C1 ŽNo. 149. and FY2A11 ŽNo. 151. in cluster T10 and FY1A3 ŽNo. 148. and FY1H2 ŽNo. 150. in
M.D. PescoÕitz et al.r Veterinary Immunology and Immunopathology 60 (1998) 251–260
255
cluster T11, were generated by immunizing mice with a peptide deduced from the sequence of porcine CDe chain ŽKirkham et al., 1996.. These mAb also stained COS cells transiently expressing the porcine CD3e chain ŽKirkham et al., 1996. ŽYang et al., 1996.. mAb BB23-8E6 ŽNo. 169. and STH164 ŽNo. 088. gave a similar binding pattern to CD3e binding mAb. BB23-8E6 also stained transfected COS cells ŽPescovitz et al., 1998b.. FY2A11, a cluster T10 mAb, completely inhibited the binding of biotinylated BB23-8E6, a T11 cluster mAb used as a standard. It is unclear why these two mAb, which bind to the same epitope, segregated into different clusters. As defined elsewhere ŽPescovitz et al., 1998a., these 2 mAb react with the CD3a epitope; FY1A3 and FY2C1 react with the CD3b epitope; FY1H2 reacts with the CD3c epitope; STH164 reacts with a different epitope from the others but because of lack of confirmation of reactivity CD3 specificity as defined by the workshop, it is designated wCD3.
5. Porcine CD4 r wCD6 Six mAb distributed to cluster T14a and T14b including standards 74-12-4 ŽNo. 173, anti-CD4., a38b2 ŽNo. 175, anti-wCD6., and b30c7 ŽNo. 184, anti-SWC2.. mAb STH293 ŽNo. 092., which precipitated a 60 kDa protein ŽAasted et al., 1998. and inhibited binding of biotinylated 74-12-4 but not anti-CD2 ŽTable 2., was assigned to CD4a. MAb STH055 ŽNo. 087. which immunoprecipitated a 50 kDa protein and did not block 74-12-4, could not be clearly assigned to CD4. It may be another anti-SWC2 and awaits further analysis. Also within this group was mAb MIL8 ŽNo. 082., which precipitated a protein of 150 kDa ŽAasted et al., 1998. which was similar to that to a38b2 but did not inhibit binding of a38b2. This mAb was therefore assigned to wCD6b ŽPescovitz et al., 1998b..
6. Porcine wCD5 Three mAb were distributed to cluster T13 including the anti-CD5 standard b53b7 ŽNo. 174.. The two new mAb 1H6r8 ŽNo. 058. and BB6-9G12 ŽNo. 166. both precipitated proteins of about 55 and 60 kDa. These two new mAb both blocked binding of b53b7 and have been assigned to the CD5a epitope ŽPescovitz et al., 1998c..
7. Porcine wCD8 Five mAb distributed to cluster T9 which included the wCD8a standard 76-2-11 ŽNo. 176. and the wCD8b standard 11r295r33 ŽNo. 177.. The three new mAb precipitated the same mw proteins of 33 and 36 kDa as did the controls. As defined elsewhere in this volume ŽZuckermann et al., 1998., mAb STH101 ŽNo. 090. was assigned to wCD8a. mAb PG164A ŽNo. 051. defined a new CD8 epitope wCD8c that is present only on the
256
M.D. PescoÕitz et al.r Veterinary Immunology and Immunopathology 60 (1998) 251–260
Table 1 Percentage binding of workshop mAb assigned to the T-cell panel Workshop number
mAb Name
014 024 032 033 034 035 036 037 038 039 040 043 044 045 046 047 048 051 057 058 061 062 082 085 086 087 088 090 092 115 119 125 131 139 145 148 149 150 151 152 154 158 160 166 168 169 171
PG107A PG104A PG139A GB16A PGB70A PGB84A PGB89A PGBL2A PGBL7A PGBL11A PGBL16A PGBL25A PGBL31A PGB95A PGBL6A PGBL13A PGBL23A PG164A 2F6r8 1H6r8 5A6r8 6H4r7 MIL8 K139.3E1 K252.3D11 STH055 STH164 STH101 STH293 1030-3-17 1030H-2-10 IAH-CC57 UCP1C10 UCP1H122 C28 FY1A3 FY2C1 FY1H2 FY2A11 FQ4G7 FA2F12 FQ3E8 FS2H10 BB6-9G12 BB6-10A10 BB23-8E6 76-7-4
Antigen assignment
wCD25
CD2a CD2a wCD8c CD5a
wCD6
wCD3 wCD8a CD4a
wCD8 CD3b CD3b CD3c CD3a
CD5a CD3a wCD1
Thymocytes
PBL
Splenocytes
Peyer’s patch
PHA Blast
33–36 0–5 0–3 0–3 0–4 0–1 0–2 0–2 0 0–5 4–7 3–5 0–3 59–70 83–93 0 81–93 51–68 0–1 49–87 0 0–2 72 0–1 7–20 54–64 37–59 55–73 59–70 0 0 0 0–1 60–75 0 41–48 18–37 53–93 17–37 1–4 0–5 0 0 68–94 0–2 38–95 59–83
28–45 6–10 0–1 0 0–3 0–4 0 0 1–10 1–10 0–6 3–4 3–8 22–34 44–76 0–3 46–66 8–21 5–6 66–72 0 0 37–57 6–14 28–32 52–70 64–73 17–24 24–41 5–9 0–8 0 0 16–24 0 64–75 57–73 68–75 59–74 0 11 0 1 63–72 3 68–79 6–10
9–63 8–47 4–12 0–4 0–48 3–19 0–5 0–4 10–18 7–44 1–30 7–32 1–2 6–100 29–57 1–16 25–43 2–14 2–11 16–49 0–15 3–21 13–46 13–39 11–20 7–48 34–47 6–43 7–15 1–3 0–2 0–2 0–1 3–42 3–19 35–45 3–29 43–55 1–34 4–22 19–45 0–2 0 26–36 4–10 42–76 12–15
5–34 1–6 2–22 0–10 0–20 1–11 0–8 0 3–17 3–17 0–3 0–9 0 0–2 8–9 0 7–10 3 0–2 4–8 0–2 0–2 2–7 4–9 2 4 8–9 3–5 1–2 0 0 0 0 3–7 0 7–9 5–9 9–15 4–8 3–22 71–82 0 0 5–8 36–45 9–76 3–17
54 3 32 0 0 61 41 95 23 4 58 93 0 0 93 35 92 21 4 93 92 0 91 2 90 84 92 15 79 0 0 3 0 34 0 ND 91 90 93 9 27 2 69 ND ND 91 92
M.D. PescoÕitz et al.r Veterinary Immunology and Immunopathology 60 (1998) 251–260
257
Table 1 Žcontinued. Workshop number
mAb Name
Antigen assignment
Thymocytes
PBL
Splenocytes
Peyer’s patch
PHA Blast
172 173 174 175 176 177 179 183 184 186
MSA4 74-12-4 b53b7 a38b2 76-2-11 11r295r33 K231.3B2 11r8r1 b30c7 MAC319
CD2a CD4a CD5a wCD6 wCD8a wCD8b wCD25 SWC1a SWC2 SWC4
84–94 65–80 47–93 67–70 45–72 61–76 03 ND 45–54 0–87
47–65 41 62–68 39–54 16–24 18–32 3–5 72 40–74 0–74
32–61 23–47 18–41 8–43 5–42 5–45 1–29 0 1–46 1–45
8–10 4–8 4–8 3–8 3–6 4–7 0–2 ND 1–2 5–62
90 82 91 67 15 23 92 6 70 0
CD8 bright cells and not the CD4rCD8 dual expressors. Two additional mAb STH106 ŽNo. 094. and SwNL554.1 ŽNo. 009. showed blocking of PG164A and seem to recognize the wCD8c epitope. This fact and also the results with UCP1H122 ŽNo. 139., which has been assigned to wCD8 without an epitope designation are still somewhat ambiguous and will be further studied in the next workshop. 8. Porcine wCD25 Two mAb distributed to the cluster T8 one of which was K231.3B2 ŽNo. 179., the standard anti-wCD25 mAb defined in the First CD Workshop ŽBailey et al., 1992.. The other mAb, PGBL25A ŽNo. 043. gave the same pattern on stimulated cell populations and precipitated a 74 kDa protein. No blocking experiments were performed and since no sequence verification is yet available, these two mAb were assigned to wCD25. Table 2 Epitope analysis of the CD2 and CD4 mAb expressed as percentage inhibition of standard anti-CD2 and CD4 mAb Inhibitor
Media PGBL6A ŽNo. 046. PGBL23A ŽNo. 048. STH055 ŽNo. 087. STH293 ŽN0. 092. MSA4 ŽNo. 172. 74-12-4 ŽNo. 173.
CD Designation
CD2 CD2 CD4 CD2 CD4
% Inhibition of CD2
CD4
0 100 100 21 8 70 8
0 23 12 6 100 24 100
Peripheral blood lymphocytes were first incubated with either media or the indicated culture supernate. Without washing, either anti-CD2 or anti-CD4 biotin conjugated mAb was added. After incubation for 30 min, the cells were washed and then stained with Streptavidin-PE. Percentage inhibition of the mean channel fluorescence was calculated with the formula Ž1-Žexperimental-background.rŽstandard-background..=100, where the standard was the biotin mAb with saline. The mean background channel of the PBL with Streptavidin-PE without biotinylated-mAb, the background, was 7.
258
M.D. PescoÕitz et al.r Veterinary Immunology and Immunopathology 60 (1998) 251–260
9. Activation mAb As opposed to the First Workshop, quite a few mAb reacting with cell surface proteins whose expression varied with activation besides CD25 were identified. Four mAb were distributed in pairs to clusters T2 and T7. Within T7 mAb PG139 ŽNo. 032. and PGBL16A ŽNo. 040. stained about half of the blasts but very few resting cells. The molecular weight of the PG139 antigen was found to be 43 kDa whereas that of the PGBL16A antigen is unknown. Cluster T2, composed of PGB84A ŽNo. 035. and PGB89A ŽNo. 036. was similar in reactivity to T7 on blasting and resting cells, and mAb PGB84A precipitated a 47 kDa protein. Expression of the PGBL16A epitope appears slightly earlier than that of the other mAb ŽTable 3.. Epitope studies were not done which would have been able to help determine if T7 and T2 contained mAb reactive with the same protein. These findings of a 43 to 47 kDa protein expressed on activated T-cells are consistent with both the CD38 and CD40 antigens. Additional work will be needed to define what the CD designation should be and to further resolve whether these mAb form a new cluster. Three clusters T1, T5, and T6 appear to contain mAb whose antigens are decreased with activation. Cluster T1 composed of 2F6r8 ŽNo. 057. and 6H4r7 ŽNo. 062. reacted with a variable percentage of resting cells but no blasts. 2F6r8 was clustered into SWC7 an antigen present on B-cells. The molecular weight of the 6H4r7 antigen was found to be 32 and 38 kDa. mAb 1030-3-17 ŽNo. 115. and 1030H-2-10 ŽNo. 119. members of T5 also only stain a few percentage of peripheral blood cells and no blasts. The molecular weight of the 1030H-2-10 antigen was found to be about 17 kDa. Finally, GB16A ŽNo. 033. and PGB70A Ž034. make up T6. GB16A appears to react with Con A and precipitated a 30 kDa protein, whereas PGB70A precipitated a 100 kDa protein ŽDavis, unpublished results.. Six other mAb did not cluster but also demonstrated activation state dependent binding. Those with increased expression on blasts and, if available, the molecular
Table 3 Kinetics of appearance of mAb-defined molecules expressed on the surface of porcine Con ArPWM stimulated lymphocytes mAb
Workshop number
Time of first detection Žh.
PGB70A PGBL16A PGBL25A PGB84A PGB89A PGBL7A PGBL11A PG139A
034 040 043 035 036 038 039 032
4 16 16 24 24 24 24 24
Peripheral blood lymphocytes from 3 animals were simultaneously stimulated in vitro in complete media with 5 m grml ConA and 2.5 m grPWM. Cells were collected at 4, 16–18, 24, and 48 h and stained for flow cytometry with several of the workshop mAb that had been defined as reactive with activation markers. The cells were then analyzed by flow cytometry.
M.D. PescoÕitz et al.r Veterinary Immunology and Immunopathology 60 (1998) 251–260
259
weight of the target antigen included: PG107A ŽNo. 014.; FA2F12 ŽNo. 154.; 5A6r8 ŽNo. 061, mw 25r30 kDa.; and FS2H10 ŽNo. 160.. Those that decreased included: 11r8r1 ŽNo. 183, the SWC1 standard.; and FQ4G7 ŽNo. 152, mw 80 kDa..
10. Summary The analysis of the T-cellractivation group of mAb provided new antibodies that were not found during the first workshop. Foremost among these are 6 mAb to CD3, and 1 mAb to a CD8 epitope, perhaps to the b chain, that appears to be present only on the CD8 bright or single positive cells. The weakest portion of the analysis continues to be those mAb that react with activation markers. This area should be a focus of more detailed analyses in the next workshop.
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. Bailey, M., Stevens, K., Bland, P.W., Stokes, C.R., 1992. A monoclonal antibody recognizing an epitope associated with pig interleukin-2 receptors. J. Immunol. Meth. 153, 85–91. Carr, M.M., Lunney, J.K., Arn, S., Aasted, B., Binns, R.M., Davis, W.C., Choi, C., Howard, C.J., Misfeldt, M., Molitor, T., Murtaugh, M., Pescovitz, M., Saalmuller, A., Sachs, D., Sopp, P., Walker, K., 1994. ¨ Summary of first round cluster analysis: complete antibody panel. Vet. Immunol. Immunopathol. 43, 211–217. Kirkham, P.A., Takamatsu, H., Yang, H., Parkhouse, R.M.E., 1996. Porcine CD3e: its characterization, expression and involvement in activation of porcine T-lymphocytes. Immunology 87, 616–623. Pescovitz, M.D., Book, B.K., Aasted, B., Dominguez, J., Ezguerra, A., Trebichavsky, I., Novikov, B., Valpotic, I., Nielsen, J., Arn, S., Sachs, D.H., Lunney, J.K., Boyd, P.C., Walker, J., Lee, R., Lackovic, G., Kirkham, P., Parkhouse, R.M.E., Saalmuller, A., 1998a. Analyses of monoclonal antibodies reacting with ¨ porcine CD3: results from the Second International Swine CD Workshop. Vet. Immunol. Immunopathol. 60, 259–266. Pescovitz, M.D., Book, B.K., Aasted, B., Dominguez, J., Bullido, R., Trebichavsky, I., Novikov, B., Valpotic, I., Nielsen, J., Arn, S., Sachs, D.H., Lunney, J.K., Boyd, P.C., Walker, J., Lee, R., Petrinec, N., Saalmuller, A., 1998b. Analyses of monoclonal antibodies reacting with porcine wCD6: results from the ¨ Second International Swine CD Workshop. Vet. Immunol. Immunopathol. 60, 273–291. Pescovitz, M.D., Book, B.K., Aasted, B., Dominguez, J., Bullido, R., Trebichavsky, I., Novikov, B., Valpotic, Tomaskovic, M., Nielsen, J., Arn, S., Sachs, D.H., Lunney, J.K., Boyd, P.C., Walker, J., Lee, R., Saalmuller, A., 1998c. Analyses of monoclonal antibodies reacting with porcine CD5: results from the ¨ Second International Swine CD Workshop. Vet. Immunol. Immunopathol. 60, 267–271. Saalmuller, A., Aasted, B., Canals, A., Dominguez, J., Goldman, T., Lunney, J.K., Mauer, S., Pescovitz, ¨ M.D., Pospisil, R., Salmon, H., Tlaskalova, H., Valpotic, I., Vizcaino, J.S., Weiland, E., Zuckermann, F., 1994. Summary of workshop findings for porcine T-lymphocytes antigens. Vet. Immunol. Immunopathol. 43, 219–228. Saalmuller, A., Pauly, T., Aasted, B., Jensen, K.T., Sachs, D.H., Arn, S., Davis, W.C., Park, Y.H., ¨ McCullough, K., Summerfield, A., Murtaugh, M., Pampusch, M.S., Burger, K.D., Laber, J., Nielsen, J., Pescovitz, M.D., Stokes, C.R., Haverson, K., Boyd, P.C., Lunney, J.K., 1998. Summary of the first round analyses of the Second International Swine CD Workshop. Vet. Immunol. Immunopathol. 60, 237–248.
260
M.D. PescoÕitz et al.r Veterinary Immunology and Immunopathology 60 (1998) 251–260
Yang, H., Oura, C.A., Kirkham, P.A., Parkhouse, R.M.E., 1996. Preparation of monoclonal anti-porcine CD3 antibodies and preliminary characterization of porcine T-lymphocytes. Immunology 88, 577–585. Zuckermann, F.A., Pescovitz, M.D., Aasted, B., Dominguez, J., Trebichavsky, I., Novikov, B., Valpotic, I., Nielsen, J., Arn, S., Sachs, D.H., Lunney, J.K., Boyd, P.C., Walker, J., Lee, R., Davis, W.C., Barbosa, I.R., Saalmuller, A., 1998. Report on the analyses of monoclonal antibodies reactive with porcine CD8 for ¨ the Second International Swine CD Workshop. Vet. Immunol. Immunopathol. 60, 299–311.
Summary of workshop findings for antibodies reacting with porcine T-cells and activation antigens: results from the Second International Swine CD Workshop M.D. Pescovitz a,) , B.K. Book a , B. Aasted b, J. Dominguez c , A. Ezquerra c , I. Trebichavsky d , B. Novikov e, I. Valpotic f , L. Sver f , J. Nielsen g , S. Arn h , D.H. Sachs h , J.K. Lunney i , P.C. Boyd i , J. Walker j, R. Lee j, William Davis k , I.R. Barbosa k , m F. Zuckermann l , A. Saalmuller ¨ a
Indiana UniÕersity, Department 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 h 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 Washington State UniÕersity, Pullman, WA, USA l Department of Veterinary Pathology, UniÕersity of Illinois, Urbana, IL, USA m Federal Research Center for Virus Diseases of Animals, Tubingen, Germany ¨
Abstract After initial evaluation of the 176 new and 19 control monoclonal antibodies ŽmAb. submitted to the Second International Swine CD Workshop, 57 were assigned to the T-cellractivation marker subgroup. These 57 mAb were further analyzed using flow cytometry on whole blood lymphocytes, splenocytes, Peyer’s patch lymphocytes, in vitro cell lines, broncho-alveolar lavage cells, Con A and PHA blasts, fetal cell populations, and by 2-color flow cytometry against mAb to
)
Corresponding 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 1 - 3
252
M.D. PescoÕitz et al.r Veterinary Immunology and Immunopathology 60 (1998) 251–260
porcine CD2, CD4, and CD8. Finally, the molecular weights of the target antigens were characterized when possible. As a result of these analyses, 23 mAb were distributed into 7 CD clusters. Newly confirmed mAb assignments included: two CD2; one CD4; two CD5; one wCD6; and one wCD25. Three new mAb were found that reacted with wCD8, one of which defined a new epitope, wCD8c. For the first time, mAb against porcine CD3 were identified, including 6 mAb that reacted with three different epitopes. Several new mAb reacted with antigens whose expression varied depending on the activation state of the test cell. These will require further characterization in order to assign a CD number. q 1998 Elsevier Science B.V. Keywords: Monoclonal antibodies; T-cells; Swine CD workshop; Porcine
1. Introduction International workshops designed to categorize into Clusters of Differentiation ŽCD. large numbers of independently generated monoclonal antibodies against lymphocyte populations, have been extremely successful. Following this pattern, the First International Swine CD Workshop confirmed the assignment of mAb available at that time to well-defined CD groups, some of which were analogous to those seen with other species, and some unique to the pig ŽCarr et al., 1994. ŽSaalmuller et al., 1994.. The ¨ success of this first workshop, and the recognized need to identify other mAb to CD described in other species, prompted the organization of the Second International Swine CD Workshop. This workshop, orchestrated by Dr. Armin Saalmuller, analyzed 176 ¨ mAb in a first round of analyses. These analyses assigned 51 of the 176 mAb to 6 clusters, which appeared to react with T-cells or activation markers ŽSaalmuller et al., ¨ 1998.. Six additional mAb, which were not assigned to these clusters but according the opinion of the submitting laboratory were T-cell reactive, were included in the subsequent analyses. These additional analyses confirmed the assignment of most of the mAb as reactive with T-cell or activation antigens and further grouped the mAb into 7 well-defined CD groups.
2. Methods Fifty-seven mAb identified as reacting with T-cells or activation markers, including 11 standards ŽSaalmuller ¨ et al., 1998., were distributed to the authors of this paper. The mAb were then tested by one, and in some cases 2 color, flow cytometry. The cell populations used for one color immunostaining included both post natal and fetal thymocytes, splenocytes, lymphocytes from mesenteric lymph nodes and Peyer’s Patches, peripheral blood mononuclear cells, white blood cells, and bone marrow cells. Various in vitro porcine cell lines and both PHA and Con A blasts were also studied. Two color Fig. 1. Dendogram of mAb analyzed by the T-cellrActivation group. The clusters that have been assigned are labeled T1 to T14b. All mAb that did not cluster with at least one other mAb are labeled ‘U’. The small numbers to the left are the assigned workshop numbers; the corresponding mAb designations from the submitting laboratories are listed in Table 1.
M.D. PescoÕitz et al.r Veterinary Immunology and Immunopathology 60 (1998) 251–260
253
254
M.D. PescoÕitz et al.r Veterinary Immunology and Immunopathology 60 (1998) 251–260
analyses were performed with the workshop mAb, counter staining with anti-pig Ig to exclude B-cells from the analysis, and against standard anti-CD2, CD4, and CD8 mAb. These analyses gave 39 data sets, which were further analyzed by the Database IV software from the Fourth Human Leukocyte Workshop. The clustering after this analysis is shown in Fig. 1. Further definition of the clusters was aided by the determination of the molecular weight of the target cell antigen ŽAasted et al., 1998.. For mAbs assigned to the clusters T12 containing the CD2 standard, and cluster T14a and T14b containing the CD4 and wCD6 standards epitope analysis was performed. Peripheral blood lymphocytes were first incubated with the workshop antibody to be tested or appropriate control antibodies. Then without washing, suboptimal amounts of either biotin conjugated anti-wCD2 or anti-wCD4 mAb were added. The cells were then washed and stained with streptavidin-PE. 3. Results The reactivity of the 57 mAb with representative cell populations is summarized in Table 1. Thirty-five mAb were grouped into 14 well-defined clusters of at least 2 mAb ŽFig. 1.. Five mAb ŽPGBL31A, No. 044; IAH-CC57, No. 125; UCP1C10, No. 131; C28, No. 145; and FQ3E8, No. 158. clustered tightly in T4 but their staining was very weak at best and the clustering may have been an artifact. Seventeen mAb Žnoted as ‘U’ in Fig. 1. did not cluster with any other mAb. These included several of the internal standards: 76-7-4 ŽNo. 171, CD1.; 11r8r1 ŽNo. 183, SWC1.; and MAC319 ŽNo. 186, SWC4.. Two mAb BB6-10A10 ŽNo. 168. and K139.3E1 ŽNo. 085. were further analyzed in the B-cell section. MAb PGBL2A ŽNo. 037. appeared to block binding of the standard anti-CD2 mAb, MSA4, but it did not cluster in T12 with the CD2 standard, and the molecular weight of the target antigen was not known. However, it is thought that this mAb is anti-Con A ŽDavis, unpublished results.. mAb PGBL13A ŽNo. 047. and PG104A ŽNo. 024. immunoprecipitated a similar molecular weight protein of 14r16 kDa ŽAasted et al., 1998. but clearly gave different reactivity patterns with intact cells. 4. Porcine CD2 Three mAb distributed to cluster T12 including the standard anti-CD2 mAb MSA4 ŽNo. 172.. The two new mAb PGBL6A ŽNo. 046. and PGBL23A ŽNo. 048. inhibited binding of biotinylated MSA4 but not anti-CD4 ŽTable 2.. In neither case was the target antigen immunoprecipitated. On the basis of on the clustering and the inhibition, however, these mAb have been assigned to CD2a. 4.1. Porcine CD3 Six monoclonal antibodies distributed into cluster T10 and T11. There were no previously identified standards with which to compare these mAb. All of the mAb precipitated a 24 kDa protein on reduced polyacrylamide gels. mAbs FY2C1 ŽNo. 149. and FY2A11 ŽNo. 151. in cluster T10 and FY1A3 ŽNo. 148. and FY1H2 ŽNo. 150. in
M.D. PescoÕitz et al.r Veterinary Immunology and Immunopathology 60 (1998) 251–260
255
cluster T11, were generated by immunizing mice with a peptide deduced from the sequence of porcine CDe chain ŽKirkham et al., 1996.. These mAb also stained COS cells transiently expressing the porcine CD3e chain ŽKirkham et al., 1996. ŽYang et al., 1996.. mAb BB23-8E6 ŽNo. 169. and STH164 ŽNo. 088. gave a similar binding pattern to CD3e binding mAb. BB23-8E6 also stained transfected COS cells ŽPescovitz et al., 1998b.. FY2A11, a cluster T10 mAb, completely inhibited the binding of biotinylated BB23-8E6, a T11 cluster mAb used as a standard. It is unclear why these two mAb, which bind to the same epitope, segregated into different clusters. As defined elsewhere ŽPescovitz et al., 1998a., these 2 mAb react with the CD3a epitope; FY1A3 and FY2C1 react with the CD3b epitope; FY1H2 reacts with the CD3c epitope; STH164 reacts with a different epitope from the others but because of lack of confirmation of reactivity CD3 specificity as defined by the workshop, it is designated wCD3.
5. Porcine CD4 r wCD6 Six mAb distributed to cluster T14a and T14b including standards 74-12-4 ŽNo. 173, anti-CD4., a38b2 ŽNo. 175, anti-wCD6., and b30c7 ŽNo. 184, anti-SWC2.. mAb STH293 ŽNo. 092., which precipitated a 60 kDa protein ŽAasted et al., 1998. and inhibited binding of biotinylated 74-12-4 but not anti-CD2 ŽTable 2., was assigned to CD4a. MAb STH055 ŽNo. 087. which immunoprecipitated a 50 kDa protein and did not block 74-12-4, could not be clearly assigned to CD4. It may be another anti-SWC2 and awaits further analysis. Also within this group was mAb MIL8 ŽNo. 082., which precipitated a protein of 150 kDa ŽAasted et al., 1998. which was similar to that to a38b2 but did not inhibit binding of a38b2. This mAb was therefore assigned to wCD6b ŽPescovitz et al., 1998b..
6. Porcine wCD5 Three mAb were distributed to cluster T13 including the anti-CD5 standard b53b7 ŽNo. 174.. The two new mAb 1H6r8 ŽNo. 058. and BB6-9G12 ŽNo. 166. both precipitated proteins of about 55 and 60 kDa. These two new mAb both blocked binding of b53b7 and have been assigned to the CD5a epitope ŽPescovitz et al., 1998c..
7. Porcine wCD8 Five mAb distributed to cluster T9 which included the wCD8a standard 76-2-11 ŽNo. 176. and the wCD8b standard 11r295r33 ŽNo. 177.. The three new mAb precipitated the same mw proteins of 33 and 36 kDa as did the controls. As defined elsewhere in this volume ŽZuckermann et al., 1998., mAb STH101 ŽNo. 090. was assigned to wCD8a. mAb PG164A ŽNo. 051. defined a new CD8 epitope wCD8c that is present only on the
256
M.D. PescoÕitz et al.r Veterinary Immunology and Immunopathology 60 (1998) 251–260
Table 1 Percentage binding of workshop mAb assigned to the T-cell panel Workshop number
mAb Name
014 024 032 033 034 035 036 037 038 039 040 043 044 045 046 047 048 051 057 058 061 062 082 085 086 087 088 090 092 115 119 125 131 139 145 148 149 150 151 152 154 158 160 166 168 169 171
PG107A PG104A PG139A GB16A PGB70A PGB84A PGB89A PGBL2A PGBL7A PGBL11A PGBL16A PGBL25A PGBL31A PGB95A PGBL6A PGBL13A PGBL23A PG164A 2F6r8 1H6r8 5A6r8 6H4r7 MIL8 K139.3E1 K252.3D11 STH055 STH164 STH101 STH293 1030-3-17 1030H-2-10 IAH-CC57 UCP1C10 UCP1H122 C28 FY1A3 FY2C1 FY1H2 FY2A11 FQ4G7 FA2F12 FQ3E8 FS2H10 BB6-9G12 BB6-10A10 BB23-8E6 76-7-4
Antigen assignment
wCD25
CD2a CD2a wCD8c CD5a
wCD6
wCD3 wCD8a CD4a
wCD8 CD3b CD3b CD3c CD3a
CD5a CD3a wCD1
Thymocytes
PBL
Splenocytes
Peyer’s patch
PHA Blast
33–36 0–5 0–3 0–3 0–4 0–1 0–2 0–2 0 0–5 4–7 3–5 0–3 59–70 83–93 0 81–93 51–68 0–1 49–87 0 0–2 72 0–1 7–20 54–64 37–59 55–73 59–70 0 0 0 0–1 60–75 0 41–48 18–37 53–93 17–37 1–4 0–5 0 0 68–94 0–2 38–95 59–83
28–45 6–10 0–1 0 0–3 0–4 0 0 1–10 1–10 0–6 3–4 3–8 22–34 44–76 0–3 46–66 8–21 5–6 66–72 0 0 37–57 6–14 28–32 52–70 64–73 17–24 24–41 5–9 0–8 0 0 16–24 0 64–75 57–73 68–75 59–74 0 11 0 1 63–72 3 68–79 6–10
9–63 8–47 4–12 0–4 0–48 3–19 0–5 0–4 10–18 7–44 1–30 7–32 1–2 6–100 29–57 1–16 25–43 2–14 2–11 16–49 0–15 3–21 13–46 13–39 11–20 7–48 34–47 6–43 7–15 1–3 0–2 0–2 0–1 3–42 3–19 35–45 3–29 43–55 1–34 4–22 19–45 0–2 0 26–36 4–10 42–76 12–15
5–34 1–6 2–22 0–10 0–20 1–11 0–8 0 3–17 3–17 0–3 0–9 0 0–2 8–9 0 7–10 3 0–2 4–8 0–2 0–2 2–7 4–9 2 4 8–9 3–5 1–2 0 0 0 0 3–7 0 7–9 5–9 9–15 4–8 3–22 71–82 0 0 5–8 36–45 9–76 3–17
54 3 32 0 0 61 41 95 23 4 58 93 0 0 93 35 92 21 4 93 92 0 91 2 90 84 92 15 79 0 0 3 0 34 0 ND 91 90 93 9 27 2 69 ND ND 91 92
M.D. PescoÕitz et al.r Veterinary Immunology and Immunopathology 60 (1998) 251–260
257
Table 1 Žcontinued. Workshop number
mAb Name
Antigen assignment
Thymocytes
PBL
Splenocytes
Peyer’s patch
PHA Blast
172 173 174 175 176 177 179 183 184 186
MSA4 74-12-4 b53b7 a38b2 76-2-11 11r295r33 K231.3B2 11r8r1 b30c7 MAC319
CD2a CD4a CD5a wCD6 wCD8a wCD8b wCD25 SWC1a SWC2 SWC4
84–94 65–80 47–93 67–70 45–72 61–76 03 ND 45–54 0–87
47–65 41 62–68 39–54 16–24 18–32 3–5 72 40–74 0–74
32–61 23–47 18–41 8–43 5–42 5–45 1–29 0 1–46 1–45
8–10 4–8 4–8 3–8 3–6 4–7 0–2 ND 1–2 5–62
90 82 91 67 15 23 92 6 70 0
CD8 bright cells and not the CD4rCD8 dual expressors. Two additional mAb STH106 ŽNo. 094. and SwNL554.1 ŽNo. 009. showed blocking of PG164A and seem to recognize the wCD8c epitope. This fact and also the results with UCP1H122 ŽNo. 139., which has been assigned to wCD8 without an epitope designation are still somewhat ambiguous and will be further studied in the next workshop. 8. Porcine wCD25 Two mAb distributed to the cluster T8 one of which was K231.3B2 ŽNo. 179., the standard anti-wCD25 mAb defined in the First CD Workshop ŽBailey et al., 1992.. The other mAb, PGBL25A ŽNo. 043. gave the same pattern on stimulated cell populations and precipitated a 74 kDa protein. No blocking experiments were performed and since no sequence verification is yet available, these two mAb were assigned to wCD25. Table 2 Epitope analysis of the CD2 and CD4 mAb expressed as percentage inhibition of standard anti-CD2 and CD4 mAb Inhibitor
Media PGBL6A ŽNo. 046. PGBL23A ŽNo. 048. STH055 ŽNo. 087. STH293 ŽN0. 092. MSA4 ŽNo. 172. 74-12-4 ŽNo. 173.
CD Designation
CD2 CD2 CD4 CD2 CD4
% Inhibition of CD2
CD4
0 100 100 21 8 70 8
0 23 12 6 100 24 100
Peripheral blood lymphocytes were first incubated with either media or the indicated culture supernate. Without washing, either anti-CD2 or anti-CD4 biotin conjugated mAb was added. After incubation for 30 min, the cells were washed and then stained with Streptavidin-PE. Percentage inhibition of the mean channel fluorescence was calculated with the formula Ž1-Žexperimental-background.rŽstandard-background..=100, where the standard was the biotin mAb with saline. The mean background channel of the PBL with Streptavidin-PE without biotinylated-mAb, the background, was 7.
258
M.D. PescoÕitz et al.r Veterinary Immunology and Immunopathology 60 (1998) 251–260
9. Activation mAb As opposed to the First Workshop, quite a few mAb reacting with cell surface proteins whose expression varied with activation besides CD25 were identified. Four mAb were distributed in pairs to clusters T2 and T7. Within T7 mAb PG139 ŽNo. 032. and PGBL16A ŽNo. 040. stained about half of the blasts but very few resting cells. The molecular weight of the PG139 antigen was found to be 43 kDa whereas that of the PGBL16A antigen is unknown. Cluster T2, composed of PGB84A ŽNo. 035. and PGB89A ŽNo. 036. was similar in reactivity to T7 on blasting and resting cells, and mAb PGB84A precipitated a 47 kDa protein. Expression of the PGBL16A epitope appears slightly earlier than that of the other mAb ŽTable 3.. Epitope studies were not done which would have been able to help determine if T7 and T2 contained mAb reactive with the same protein. These findings of a 43 to 47 kDa protein expressed on activated T-cells are consistent with both the CD38 and CD40 antigens. Additional work will be needed to define what the CD designation should be and to further resolve whether these mAb form a new cluster. Three clusters T1, T5, and T6 appear to contain mAb whose antigens are decreased with activation. Cluster T1 composed of 2F6r8 ŽNo. 057. and 6H4r7 ŽNo. 062. reacted with a variable percentage of resting cells but no blasts. 2F6r8 was clustered into SWC7 an antigen present on B-cells. The molecular weight of the 6H4r7 antigen was found to be 32 and 38 kDa. mAb 1030-3-17 ŽNo. 115. and 1030H-2-10 ŽNo. 119. members of T5 also only stain a few percentage of peripheral blood cells and no blasts. The molecular weight of the 1030H-2-10 antigen was found to be about 17 kDa. Finally, GB16A ŽNo. 033. and PGB70A Ž034. make up T6. GB16A appears to react with Con A and precipitated a 30 kDa protein, whereas PGB70A precipitated a 100 kDa protein ŽDavis, unpublished results.. Six other mAb did not cluster but also demonstrated activation state dependent binding. Those with increased expression on blasts and, if available, the molecular
Table 3 Kinetics of appearance of mAb-defined molecules expressed on the surface of porcine Con ArPWM stimulated lymphocytes mAb
Workshop number
Time of first detection Žh.
PGB70A PGBL16A PGBL25A PGB84A PGB89A PGBL7A PGBL11A PG139A
034 040 043 035 036 038 039 032
4 16 16 24 24 24 24 24
Peripheral blood lymphocytes from 3 animals were simultaneously stimulated in vitro in complete media with 5 m grml ConA and 2.5 m grPWM. Cells were collected at 4, 16–18, 24, and 48 h and stained for flow cytometry with several of the workshop mAb that had been defined as reactive with activation markers. The cells were then analyzed by flow cytometry.
M.D. PescoÕitz et al.r Veterinary Immunology and Immunopathology 60 (1998) 251–260
259
weight of the target antigen included: PG107A ŽNo. 014.; FA2F12 ŽNo. 154.; 5A6r8 ŽNo. 061, mw 25r30 kDa.; and FS2H10 ŽNo. 160.. Those that decreased included: 11r8r1 ŽNo. 183, the SWC1 standard.; and FQ4G7 ŽNo. 152, mw 80 kDa..
10. Summary The analysis of the T-cellractivation group of mAb provided new antibodies that were not found during the first workshop. Foremost among these are 6 mAb to CD3, and 1 mAb to a CD8 epitope, perhaps to the b chain, that appears to be present only on the CD8 bright or single positive cells. The weakest portion of the analysis continues to be those mAb that react with activation markers. This area should be a focus of more detailed analyses in the next workshop.
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. Bailey, M., Stevens, K., Bland, P.W., Stokes, C.R., 1992. A monoclonal antibody recognizing an epitope associated with pig interleukin-2 receptors. J. Immunol. Meth. 153, 85–91. Carr, M.M., Lunney, J.K., Arn, S., Aasted, B., Binns, R.M., Davis, W.C., Choi, C., Howard, C.J., Misfeldt, M., Molitor, T., Murtaugh, M., Pescovitz, M., Saalmuller, A., Sachs, D., Sopp, P., Walker, K., 1994. ¨ Summary of first round cluster analysis: complete antibody panel. Vet. Immunol. Immunopathol. 43, 211–217. Kirkham, P.A., Takamatsu, H., Yang, H., Parkhouse, R.M.E., 1996. Porcine CD3e: its characterization, expression and involvement in activation of porcine T-lymphocytes. Immunology 87, 616–623. Pescovitz, M.D., Book, B.K., Aasted, B., Dominguez, J., Ezguerra, A., Trebichavsky, I., Novikov, B., Valpotic, I., Nielsen, J., Arn, S., Sachs, D.H., Lunney, J.K., Boyd, P.C., Walker, J., Lee, R., Lackovic, G., Kirkham, P., Parkhouse, R.M.E., Saalmuller, A., 1998a. Analyses of monoclonal antibodies reacting with ¨ porcine CD3: results from the Second International Swine CD Workshop. Vet. Immunol. Immunopathol. 60, 259–266. Pescovitz, M.D., Book, B.K., Aasted, B., Dominguez, J., Bullido, R., Trebichavsky, I., Novikov, B., Valpotic, I., Nielsen, J., Arn, S., Sachs, D.H., Lunney, J.K., Boyd, P.C., Walker, J., Lee, R., Petrinec, N., Saalmuller, A., 1998b. Analyses of monoclonal antibodies reacting with porcine wCD6: results from the ¨ Second International Swine CD Workshop. Vet. Immunol. Immunopathol. 60, 273–291. Pescovitz, M.D., Book, B.K., Aasted, B., Dominguez, J., Bullido, R., Trebichavsky, I., Novikov, B., Valpotic, Tomaskovic, M., Nielsen, J., Arn, S., Sachs, D.H., Lunney, J.K., Boyd, P.C., Walker, J., Lee, R., Saalmuller, A., 1998c. Analyses of monoclonal antibodies reacting with porcine CD5: results from the ¨ Second International Swine CD Workshop. Vet. Immunol. Immunopathol. 60, 267–271. Saalmuller, A., Aasted, B., Canals, A., Dominguez, J., Goldman, T., Lunney, J.K., Mauer, S., Pescovitz, ¨ M.D., Pospisil, R., Salmon, H., Tlaskalova, H., Valpotic, I., Vizcaino, J.S., Weiland, E., Zuckermann, F., 1994. Summary of workshop findings for porcine T-lymphocytes antigens. Vet. Immunol. Immunopathol. 43, 219–228. Saalmuller, A., Pauly, T., Aasted, B., Jensen, K.T., Sachs, D.H., Arn, S., Davis, W.C., Park, Y.H., ¨ McCullough, K., Summerfield, A., Murtaugh, M., Pampusch, M.S., Burger, K.D., Laber, J., Nielsen, J., Pescovitz, M.D., Stokes, C.R., Haverson, K., Boyd, P.C., Lunney, J.K., 1998. Summary of the first round analyses of the Second International Swine CD Workshop. Vet. Immunol. Immunopathol. 60, 237–248.
260
M.D. PescoÕitz et al.r Veterinary Immunology and Immunopathology 60 (1998) 251–260
Yang, H., Oura, C.A., Kirkham, P.A., Parkhouse, R.M.E., 1996. Preparation of monoclonal anti-porcine CD3 antibodies and preliminary characterization of porcine T-lymphocytes. Immunology 88, 577–585. Zuckermann, F.A., Pescovitz, M.D., Aasted, B., Dominguez, J., Trebichavsky, I., Novikov, B., Valpotic, I., Nielsen, J., Arn, S., Sachs, D.H., Lunney, J.K., Boyd, P.C., Walker, J., Lee, R., Davis, W.C., Barbosa, I.R., Saalmuller, A., 1998. Report on the analyses of monoclonal antibodies reactive with porcine CD8 for ¨ the Second International Swine CD Workshop. Vet. Immunol. Immunopathol. 60, 299–311.