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Analyses of mAb reactive with porcine CD8
Veterinary immunology and immunopathology ELSEV I Ell
Veterinary Immunology and Immunopathology 43 (1994) 249-254
Analyses of mAb reactive with porcine CD8 A. Saalmiiller a,*, B. Aasted b, A. Canals c, J. Dominguez c, T. Goldman d, J.K. Lunney d, S. Maurer a, M.D. Pescovitz e, R. Pospisil f, H. Salmon g, I. Trebichavsky f, I. Valpotic h, J.S. Vizcaino c, F. Zuckermann i aFederalResearch Centre for Virus Diseases of Animals, P.O. Box 1149, 72001 Tiibingen, Germany bDepartment of Veterinary Virology and Immunology, Royal Veterinary University, 1870 Frederiksberg C, Denmark CDepartment de Sanidad Animal, 1NIA, 28012 Madrid, Spain dUSDA, ARS, Helminthic Diseases Laboratory, Behsville, MD 20705, USA eDepartment of Surgery arm Microbiology~Immunology, Indiana University, Indianapolis, IN 46202, USA fDepartment of Immunology, Institute of Microbiology, 14220 Prague, Czech Republic glNRA, Laboratoire dTmmunologie, Nouzilly, 37380 Monnaie, France hDepartment of Biology, Veterinary Faculty, University of Zagreb, 41000 Zagreb, Croatia iDepartment of Veterinary Pathobiolgy, College of Veterinary Medicine, Urbana, IL 61801, USA
Abstract Among all mAb submitted to the first porcine CD workshop, based on FCM analyses six mAb could be identified to recognize the porcine CD8 analogue (workshop Nos. 004, 051,052, 053, 108 and 109). In immunoprecipitation studies three mAb (Nos. 004, 108 and 109) recognized an antigen with an apparent molecular mass of about 35 kDa under reducing conditions and about 70 kDa under non-reducing conditions. The molecular masses of the antigens recognized by the three other mAb (Nos. 051,052 and 053) are still unknown. Epitope analyses performed by blocking experiments led to the determination of two CD8 epitopes: CD8a and CD8b. CD8a is recognized by mAb Nos. 004, 051 and 052, and CD8b by Nos. 053, 108 and 109.
* Corresponding author. 0165-2427/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-2427 ( 94 ) 06032-U
250
A. Saalmiiller et al. / Veterinary Immunology and lmmunopathology 43 (1994) 249-254
Table 1 Monoclonal antibodies recognizing the porcine CD8 Workshop no.
mAb
Origin/donor
Species/ isotype
Molecular mass of antigen kDa R/NR
004 051 052 053 108 109
76-2-11 PT36B PT81B PG93A 11/295/33 11/122/28
Lunney, Beltsville, MD Davis, Pullman, WA Davis, Pullman, WA Davis, Pullman, WA Saalmiiller, Tfibingen Saalmtiller, Ttibingen
mlgG2a mlgG 1 mlgG2b mlgGM mlgG2a mlgG2a
35/70 n.d. n.d. n.d. 35/70 35/70
n.d., Not determined.
1. Porcine CD8 antigens In all species studied to date, CD8 molecules are expressed on MHC I restricted T lymphocytes, which represents the predominant T-lymphocyte subset with cytolytic function. The CD8 antigens of various species are described as dimeric molecules with an apparent molecular mass of 30-35 kDa under reducing conditions and about 70 kDa under non-reducing conditions (Engleman et al., 1981 ; Ledbetter et al., 1981; Thomas and Green, 1983; Maddox et al., 1985; Ellis et al., 1986). CD8 molecules can exist as homodimers as well as heterodimers. Functionally, there is evidence that CD8 molecules interact during antigen recognition with non-polymorphic parts of MHC I molecules and enhance the avidity of binding to target cells. Three mAb--76-2-11 (No. 004), 11/295/33 (No. 108) and 11/122/28 (No. 109)--reactive with the porcine CD8 analogue have been described in 1984 (Jonjic and Koszinowski, 1984; Pescovitz et al. 1984). In immunoprecipitation studies all three mAb recognized glycosylated molecules with an apparent molecular mass of about 35 kDa under reducing conditions and about 70 kDa under non-reducing conditions. In functional studies, these mAb were able to block efficiently T lymphocytes in cell-mediated lysis of allogeneic target cells. Besides these, three other mAb--PT36B (No. 051 ), PT81B (No. 052) and PG93A (No. 053 )--with putative specificity for porcine CD8 were detected during the workshop in a common cluster (T5). All mAb with anti-CD8 reactivity are summarized in Table 1.
2. CD8 epitopes MAb-blocking and two-colour FCM analyses were used for the characterization of the epitopes recognized by the six different anti-CD8 mAb. The results confirmed for the mAb with uncharacterized molecular mass of their respective antigens (PT36B (No. 051 ), PT81B (No. 052) and PG93A (No. 053)) their CD8 reactivity. They also led to the definition of two CD8 epitopes: CD8a and
A. Saalmiiller et al. / Veterinary Immunology and Immunopathology 43 (1994) 249-254
251
10 4
._
C
10 0
101
102
10 2
10 3
10 4
fluorescence intensity
CD8a
CD8b
PT81B
#052
PG93A
76-2-11
#004
11/295/33
#053 #108
PT36B
#051
11/122/28
#109
Fig. 1. Characterization of CD8 epitopes. Porcine PBTL were labelled with different anti-CD8 mAb shown in Table 1 and an FITC-conjugated goat anti-mouse antiserum; after blocking of free binding sites of the antiserum with mouse Ig, cells were stained with biotinylated 11 / 295 / 33 (No. 108 ) and Phycoerythrin (PE)-conjugated streptavidin. The costaining ofanti-CD8a mAb with 11/295/33 (No. 108 ) is demonstrated on the left contour plot, showing representatively for mAb PT81B (No. 052 ), (FITC, abscissa ) the staining pattern of the anti-CD8a mAb, indicated in the lower part of Fig. 2, in combination with 11/295/33 (No. 108 ) (PE, ordinate). Blocking of biotinylated 11/295/33 (No. 108 ) by mAb belonging to the CD8b subcluster is displayed on the right contour plot. Preincubation with the respective mAb indicated below led to an elimination of the 11/295/33-specific PE fluorescence (ordinate).
CD8b. Fig. 1 demonstrates the blocking behaviour of the mAb belonging to the respective subsets. The CD8a epitope was recognized by mAb 76-2-11 (No. 004 ), PT36B (No. 051 ) and PT81B (No. 052). These mAb were not able to block the binding of biotinylated mAb 11/295/33 (No. 108), specific for the CD8b epitope (Fig. 1, left). MAb belonging to the CD8b subcluster--PG93A (No. 053), 11/295/33 (No. 108)and 11/122/28 (No. 109)--blocked binding ofbiotinylated 11/295/33 (No. 108) (Fig. 1, right, abscissa). Experiments employing biotinylated mAb 76-2-11 (No. 004), led to inverse results (data not shown). Whether both epitopes CD8a and CD8b are located on the same chain or on different chains of the CD8 dimer is not clear and remains to be elucidated.
3. Expression of porcine CD8 antigens Porcine CD8 antigens are highly T-cell specific; neither B lymphocytes nor cells of the myeloic lineage show any CD8 expression. In the porcine thymic and extrathymic T-lymphocyte compartment, CD8 molecules are expressed on the majority of thymocytes (Table 2, 54-84%; Fig. 2, 66%; labelling with 11/295/33 (No. 108)) and a substantial proportion of peripheral T lymphocytes in blood (Table 2, peripheral blood T lymphocytes, PBTL 28-82%; Fig. 2, 56%) and in
252
A. Saalmiiller et al. / Veterinary Immunology and lmmunopathology 43 (1994) 249-254
Table 2 Percentage of cells stained by anti-CD8 monoclonal antibodies
Workshop
mAb
Percentage positive cells in
no,
004 051 052 053 108 109
76-2-11 PT36B PT81B PG93A 11/295/33 11/122/28
Thymocytes
PBTL
SPTL
LNTL
54-75 60-78 57-76 56-77 60-84 56-78
28-53 34-79 32-79 30-79 30-82 31-79
43-77 71-77 60-81 59-73 50-80 62-80
42-45 43-79 47-53 45-54 43-52 44-54
The data presented are based at least on FCM analyses of three individual animals. PBTL, SPTL and LNTL designate nylon-wool purified T lymphocytes from peripheral blood, spleen and mesenteric lymph nodes, respectively.
thymocytes
PBTL
LNTL
35 "6 E
21 34
i
44
c . . . .
]
fluorescence intensity
CD8 Fig. 2. Expression of CD8 on thymocytes and extrathymic T lymphocytes. Cells were labelled with biotinylated mAb 11/295/33 (No. 108), and CY5-conjugated streptavidin. Horizontal bars indicate the percentage of cells with the respective fluorescence intensity.
lymphoid tissues (T lymphocytes from spleen, SPTL and mesenteric lymph nodes, LNTL, 43-80% and 42-79%, respectively; Fig. 2, LNTL, 34%). The percentages of CD8-positive cells in T-lymphocyte populations show high variations between individuals (Saalmiiller et al., 1987). Studies from different laboratories indicated that the proportion of CD8-positive cells in the extrathymic T-lymphocyte compartment is age dependent and increases with the age of the individual pig. Besides these differences in the proportion of CD8-positive cells, there is also a variation among the CD8-positive T-lymphocyte subpopulation characterized by different CD8 antigen density. In contrast to most of the other species analysed so far, porcine CD8 on extrathymic T lymphocytes shows a heterogeneous antigen expression. Both a population with high CD8 antigen density (demonstrated for one individual in Fig. 2, PBTL, 35%) and a population with low CD8 expression could be detected (Fig. 2, PBTL, 21%). Whereas the CD8-positive T-lymphocyte subset with high CD8 antigen density includes the MHC class I-restricted cytolytic T lymphocytes, the CD8 low extrathymic T-lymphocyte subset could be either identified as cells with spontaneous cytolytic activity against tum-
A. Saalmiiller et aL / Veterinary Immunology and lmmunopathology 43 (1994) 249-254
10 26
55 45
104114
140/ 10 o
LNTL
PBTL
thymocytes
101
102
103
100
253
j ,,~.
fluorescenceintensity
50 lO 0
lO 1
1o 2
103
104
CD4 Fig. 3. Coexpression of CD4 and CD8 antigens on cells from lymphoid tissues. Thymocytes, PBTL and LNTL were labelled in a two-step procedure with mAb 74-12-4 (anti-CD4, lgG2b) and biotinylated 11/295/33 (anti-CD8, IgG2a) in the first step and the specific fluorochrome labelled reagents FITC-conjugated anti-mouse IgG2b and Streptavidin-Cy5 in the second step. The CD4 vs. CD8 expression is shown as contour plots for the respective cell populations. Based on the negative controis, the contour plots were divided into four quadrants, containing double negative cells (quadrant Ill), single positive cells (quadrants I and IV) and double positive cell populations (quadrant II). Numbers in the corners of the quadrants give the percentages of the portions of the respective subpopulations.
our cells showing the in vitro function of natural-killer (NK) cells (Saalmiiller et al., 1994) or, in case of coexpression of the CD4 antigen in two-colour FCM analyses, as CD4+CD8 + double positive extrathymic T lymphocytes with Thelper activity (Fig. 3, PBTL, 10%; Pescovitz et al., 1994). This extrathymic double positive T-lymphocyte subpopulation is unique for swine and could be distinguished from double positive thymocytes (Fig. 3 ) by the lack of the thymocytespecific CD 1 antigen (Saalmfiller et al., 1989 ).
4. Summary and conclusions Based on FCM analyses, six mAb---76-2-11 (No. 004), PT36B (No. 051 ), PT81B (No. 052), PG93A (No. 053), 11/295/33 (No. 108) and 11/122/28 (No. 109)--recognizing the porcine CD8 antigen were identified to cluster together in the T5 cluster. Epitope analyses using biotinylated mAb 11/295/33 (No. 108) and 76-2-11 (No. 004) led to the determination of two epitopes CD8a and CD8b, the first recognized by mAb 76-2-11 (No. 004), PT36B (No. 051 ) and PT81B (No. 052), and the second by mAb PG93A (No. 053), 11/295/33 (No. 108) and 11 / 122/28 (No. 109). Because of missing data about the molecular mass of the antigen recognized by mAb PT36B (No. 051), PT8 IB (No. 052) and PG93A (No. 053), these mAb received the prefix 'w' for their respective subcluster.
254
A. Saalmiiller et al. / Veterinary Immunology and hnmunopathology 43 (1994) 249-254
References J.A. Ellis, C.L. Baldwin, N.D. MacHugh, A. Bensaid, A.J. Teale, B.M. Goddeeris and W.I. Morrison. 1986. Immunology 58, 351. E.G. Engleman, C.J. Benike, E. Glickman and R.L. Evans, 1981. J. Exp. Med. 153, 193. S. Jonjic and U.H. Koszinowski, 1984. J. Immunol. 133,647. J.A. Ledbetter, R.L. Evans, M. Lipinski, C. Cunningham-Rundles, R.A. Good and L.A. Herzenberg, 1981. J. Exp. Med. 153, 310. J.F. Maddox, C.R. Mackay and M.R. Brandon, 1985. Immunology 55,739. M.D. Pescovitz, J.K. Lunney and D.H. Sachs, 1984. J. Immunol. 133,368. M.D. Pescovitz, A. Sakopoulos, J.A. Gaddy, R.J. Hussman and F.A. Zuckermann, 1994. Vet. lmmunol. lmmunopathol. A. Saalmfiller, M.J. Reddehase, H.-J. Bfihring, S. Jonjic and U.H. Koszinowski, 1987. Eur. J. lmmunol. 17, 1297. A. Saalmfiller, W. Hirt and M.J. Reddehase, 1989. Eur. J. Immunol. 19, 2011. A. Saalmiiller, W. Hirt, S. Maurer, E. Weiland, 1994. Immunology 81,578. M.L. Thomas and J.R. Green, 1983. Eur. J. Immunol. 13,855.
Veterinary Immunology and Immunopathology 43 (1994) 249-254
Analyses of mAb reactive with porcine CD8 A. Saalmiiller a,*, B. Aasted b, A. Canals c, J. Dominguez c, T. Goldman d, J.K. Lunney d, S. Maurer a, M.D. Pescovitz e, R. Pospisil f, H. Salmon g, I. Trebichavsky f, I. Valpotic h, J.S. Vizcaino c, F. Zuckermann i aFederalResearch Centre for Virus Diseases of Animals, P.O. Box 1149, 72001 Tiibingen, Germany bDepartment of Veterinary Virology and Immunology, Royal Veterinary University, 1870 Frederiksberg C, Denmark CDepartment de Sanidad Animal, 1NIA, 28012 Madrid, Spain dUSDA, ARS, Helminthic Diseases Laboratory, Behsville, MD 20705, USA eDepartment of Surgery arm Microbiology~Immunology, Indiana University, Indianapolis, IN 46202, USA fDepartment of Immunology, Institute of Microbiology, 14220 Prague, Czech Republic glNRA, Laboratoire dTmmunologie, Nouzilly, 37380 Monnaie, France hDepartment of Biology, Veterinary Faculty, University of Zagreb, 41000 Zagreb, Croatia iDepartment of Veterinary Pathobiolgy, College of Veterinary Medicine, Urbana, IL 61801, USA
Abstract Among all mAb submitted to the first porcine CD workshop, based on FCM analyses six mAb could be identified to recognize the porcine CD8 analogue (workshop Nos. 004, 051,052, 053, 108 and 109). In immunoprecipitation studies three mAb (Nos. 004, 108 and 109) recognized an antigen with an apparent molecular mass of about 35 kDa under reducing conditions and about 70 kDa under non-reducing conditions. The molecular masses of the antigens recognized by the three other mAb (Nos. 051,052 and 053) are still unknown. Epitope analyses performed by blocking experiments led to the determination of two CD8 epitopes: CD8a and CD8b. CD8a is recognized by mAb Nos. 004, 051 and 052, and CD8b by Nos. 053, 108 and 109.
* Corresponding author. 0165-2427/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-2427 ( 94 ) 06032-U
250
A. Saalmiiller et al. / Veterinary Immunology and lmmunopathology 43 (1994) 249-254
Table 1 Monoclonal antibodies recognizing the porcine CD8 Workshop no.
mAb
Origin/donor
Species/ isotype
Molecular mass of antigen kDa R/NR
004 051 052 053 108 109
76-2-11 PT36B PT81B PG93A 11/295/33 11/122/28
Lunney, Beltsville, MD Davis, Pullman, WA Davis, Pullman, WA Davis, Pullman, WA Saalmiiller, Tfibingen Saalmtiller, Ttibingen
mlgG2a mlgG 1 mlgG2b mlgGM mlgG2a mlgG2a
35/70 n.d. n.d. n.d. 35/70 35/70
n.d., Not determined.
1. Porcine CD8 antigens In all species studied to date, CD8 molecules are expressed on MHC I restricted T lymphocytes, which represents the predominant T-lymphocyte subset with cytolytic function. The CD8 antigens of various species are described as dimeric molecules with an apparent molecular mass of 30-35 kDa under reducing conditions and about 70 kDa under non-reducing conditions (Engleman et al., 1981 ; Ledbetter et al., 1981; Thomas and Green, 1983; Maddox et al., 1985; Ellis et al., 1986). CD8 molecules can exist as homodimers as well as heterodimers. Functionally, there is evidence that CD8 molecules interact during antigen recognition with non-polymorphic parts of MHC I molecules and enhance the avidity of binding to target cells. Three mAb--76-2-11 (No. 004), 11/295/33 (No. 108) and 11/122/28 (No. 109)--reactive with the porcine CD8 analogue have been described in 1984 (Jonjic and Koszinowski, 1984; Pescovitz et al. 1984). In immunoprecipitation studies all three mAb recognized glycosylated molecules with an apparent molecular mass of about 35 kDa under reducing conditions and about 70 kDa under non-reducing conditions. In functional studies, these mAb were able to block efficiently T lymphocytes in cell-mediated lysis of allogeneic target cells. Besides these, three other mAb--PT36B (No. 051 ), PT81B (No. 052) and PG93A (No. 053 )--with putative specificity for porcine CD8 were detected during the workshop in a common cluster (T5). All mAb with anti-CD8 reactivity are summarized in Table 1.
2. CD8 epitopes MAb-blocking and two-colour FCM analyses were used for the characterization of the epitopes recognized by the six different anti-CD8 mAb. The results confirmed for the mAb with uncharacterized molecular mass of their respective antigens (PT36B (No. 051 ), PT81B (No. 052) and PG93A (No. 053)) their CD8 reactivity. They also led to the definition of two CD8 epitopes: CD8a and
A. Saalmiiller et al. / Veterinary Immunology and Immunopathology 43 (1994) 249-254
251
10 4
._
C
10 0
101
102
10 2
10 3
10 4
fluorescence intensity
CD8a
CD8b
PT81B
#052
PG93A
76-2-11
#004
11/295/33
#053 #108
PT36B
#051
11/122/28
#109
Fig. 1. Characterization of CD8 epitopes. Porcine PBTL were labelled with different anti-CD8 mAb shown in Table 1 and an FITC-conjugated goat anti-mouse antiserum; after blocking of free binding sites of the antiserum with mouse Ig, cells were stained with biotinylated 11 / 295 / 33 (No. 108 ) and Phycoerythrin (PE)-conjugated streptavidin. The costaining ofanti-CD8a mAb with 11/295/33 (No. 108 ) is demonstrated on the left contour plot, showing representatively for mAb PT81B (No. 052 ), (FITC, abscissa ) the staining pattern of the anti-CD8a mAb, indicated in the lower part of Fig. 2, in combination with 11/295/33 (No. 108 ) (PE, ordinate). Blocking of biotinylated 11/295/33 (No. 108 ) by mAb belonging to the CD8b subcluster is displayed on the right contour plot. Preincubation with the respective mAb indicated below led to an elimination of the 11/295/33-specific PE fluorescence (ordinate).
CD8b. Fig. 1 demonstrates the blocking behaviour of the mAb belonging to the respective subsets. The CD8a epitope was recognized by mAb 76-2-11 (No. 004 ), PT36B (No. 051 ) and PT81B (No. 052). These mAb were not able to block the binding of biotinylated mAb 11/295/33 (No. 108), specific for the CD8b epitope (Fig. 1, left). MAb belonging to the CD8b subcluster--PG93A (No. 053), 11/295/33 (No. 108)and 11/122/28 (No. 109)--blocked binding ofbiotinylated 11/295/33 (No. 108) (Fig. 1, right, abscissa). Experiments employing biotinylated mAb 76-2-11 (No. 004), led to inverse results (data not shown). Whether both epitopes CD8a and CD8b are located on the same chain or on different chains of the CD8 dimer is not clear and remains to be elucidated.
3. Expression of porcine CD8 antigens Porcine CD8 antigens are highly T-cell specific; neither B lymphocytes nor cells of the myeloic lineage show any CD8 expression. In the porcine thymic and extrathymic T-lymphocyte compartment, CD8 molecules are expressed on the majority of thymocytes (Table 2, 54-84%; Fig. 2, 66%; labelling with 11/295/33 (No. 108)) and a substantial proportion of peripheral T lymphocytes in blood (Table 2, peripheral blood T lymphocytes, PBTL 28-82%; Fig. 2, 56%) and in
252
A. Saalmiiller et al. / Veterinary Immunology and lmmunopathology 43 (1994) 249-254
Table 2 Percentage of cells stained by anti-CD8 monoclonal antibodies
Workshop
mAb
Percentage positive cells in
no,
004 051 052 053 108 109
76-2-11 PT36B PT81B PG93A 11/295/33 11/122/28
Thymocytes
PBTL
SPTL
LNTL
54-75 60-78 57-76 56-77 60-84 56-78
28-53 34-79 32-79 30-79 30-82 31-79
43-77 71-77 60-81 59-73 50-80 62-80
42-45 43-79 47-53 45-54 43-52 44-54
The data presented are based at least on FCM analyses of three individual animals. PBTL, SPTL and LNTL designate nylon-wool purified T lymphocytes from peripheral blood, spleen and mesenteric lymph nodes, respectively.
thymocytes
PBTL
LNTL
35 "6 E
21 34
i
44
c . . . .
]
fluorescence intensity
CD8 Fig. 2. Expression of CD8 on thymocytes and extrathymic T lymphocytes. Cells were labelled with biotinylated mAb 11/295/33 (No. 108), and CY5-conjugated streptavidin. Horizontal bars indicate the percentage of cells with the respective fluorescence intensity.
lymphoid tissues (T lymphocytes from spleen, SPTL and mesenteric lymph nodes, LNTL, 43-80% and 42-79%, respectively; Fig. 2, LNTL, 34%). The percentages of CD8-positive cells in T-lymphocyte populations show high variations between individuals (Saalmiiller et al., 1987). Studies from different laboratories indicated that the proportion of CD8-positive cells in the extrathymic T-lymphocyte compartment is age dependent and increases with the age of the individual pig. Besides these differences in the proportion of CD8-positive cells, there is also a variation among the CD8-positive T-lymphocyte subpopulation characterized by different CD8 antigen density. In contrast to most of the other species analysed so far, porcine CD8 on extrathymic T lymphocytes shows a heterogeneous antigen expression. Both a population with high CD8 antigen density (demonstrated for one individual in Fig. 2, PBTL, 35%) and a population with low CD8 expression could be detected (Fig. 2, PBTL, 21%). Whereas the CD8-positive T-lymphocyte subset with high CD8 antigen density includes the MHC class I-restricted cytolytic T lymphocytes, the CD8 low extrathymic T-lymphocyte subset could be either identified as cells with spontaneous cytolytic activity against tum-
A. Saalmiiller et aL / Veterinary Immunology and lmmunopathology 43 (1994) 249-254
10 26
55 45
104114
140/ 10 o
LNTL
PBTL
thymocytes
101
102
103
100
253
j ,,~.
fluorescenceintensity
50 lO 0
lO 1
1o 2
103
104
CD4 Fig. 3. Coexpression of CD4 and CD8 antigens on cells from lymphoid tissues. Thymocytes, PBTL and LNTL were labelled in a two-step procedure with mAb 74-12-4 (anti-CD4, lgG2b) and biotinylated 11/295/33 (anti-CD8, IgG2a) in the first step and the specific fluorochrome labelled reagents FITC-conjugated anti-mouse IgG2b and Streptavidin-Cy5 in the second step. The CD4 vs. CD8 expression is shown as contour plots for the respective cell populations. Based on the negative controis, the contour plots were divided into four quadrants, containing double negative cells (quadrant Ill), single positive cells (quadrants I and IV) and double positive cell populations (quadrant II). Numbers in the corners of the quadrants give the percentages of the portions of the respective subpopulations.
our cells showing the in vitro function of natural-killer (NK) cells (Saalmiiller et al., 1994) or, in case of coexpression of the CD4 antigen in two-colour FCM analyses, as CD4+CD8 + double positive extrathymic T lymphocytes with Thelper activity (Fig. 3, PBTL, 10%; Pescovitz et al., 1994). This extrathymic double positive T-lymphocyte subpopulation is unique for swine and could be distinguished from double positive thymocytes (Fig. 3 ) by the lack of the thymocytespecific CD 1 antigen (Saalmfiller et al., 1989 ).
4. Summary and conclusions Based on FCM analyses, six mAb---76-2-11 (No. 004), PT36B (No. 051 ), PT81B (No. 052), PG93A (No. 053), 11/295/33 (No. 108) and 11/122/28 (No. 109)--recognizing the porcine CD8 antigen were identified to cluster together in the T5 cluster. Epitope analyses using biotinylated mAb 11/295/33 (No. 108) and 76-2-11 (No. 004) led to the determination of two epitopes CD8a and CD8b, the first recognized by mAb 76-2-11 (No. 004), PT36B (No. 051 ) and PT81B (No. 052), and the second by mAb PG93A (No. 053), 11/295/33 (No. 108) and 11 / 122/28 (No. 109). Because of missing data about the molecular mass of the antigen recognized by mAb PT36B (No. 051), PT8 IB (No. 052) and PG93A (No. 053), these mAb received the prefix 'w' for their respective subcluster.
254
A. Saalmiiller et al. / Veterinary Immunology and hnmunopathology 43 (1994) 249-254
References J.A. Ellis, C.L. Baldwin, N.D. MacHugh, A. Bensaid, A.J. Teale, B.M. Goddeeris and W.I. Morrison. 1986. Immunology 58, 351. E.G. Engleman, C.J. Benike, E. Glickman and R.L. Evans, 1981. J. Exp. Med. 153, 193. S. Jonjic and U.H. Koszinowski, 1984. J. Immunol. 133,647. J.A. Ledbetter, R.L. Evans, M. Lipinski, C. Cunningham-Rundles, R.A. Good and L.A. Herzenberg, 1981. J. Exp. Med. 153, 310. J.F. Maddox, C.R. Mackay and M.R. Brandon, 1985. Immunology 55,739. M.D. Pescovitz, J.K. Lunney and D.H. Sachs, 1984. J. Immunol. 133,368. M.D. Pescovitz, A. Sakopoulos, J.A. Gaddy, R.J. Hussman and F.A. Zuckermann, 1994. Vet. lmmunol. lmmunopathol. A. Saalmfiller, M.J. Reddehase, H.-J. Bfihring, S. Jonjic and U.H. Koszinowski, 1987. Eur. J. lmmunol. 17, 1297. A. Saalmfiller, W. Hirt and M.J. Reddehase, 1989. Eur. J. Immunol. 19, 2011. A. Saalmiiller, W. Hirt, S. Maurer, E. Weiland, 1994. Immunology 81,578. M.L. Thomas and J.R. Green, 1983. Eur. J. Immunol. 13,855.