Scand. J. Immunol, 36, Suppl. 11, 199-201, 1992
Reactivity of some Monoclonal Antibodies Specific for Human Lymphocytes with Vervet Monkey Peripheral Blood Mononuclear Cells J. O. OLOBO Institute of Primate Research, National Museums of Kenya, Nairobi, Kenya
Olobo JO. Reactivity of some Monoclonal Antibodies Specific for Human Lymphocytes with Vervet Monkey Peripheral Blood Mononuclear Cells. Scand J Immunol 1992;36(Suppl. 11):I99201 Reactivities of some monoclonal antibodies to human lymphocyte surface antigens were tested on vervet monkey peripheral blood mononuclear cells (PBMC), using flow cytometry and immunoperoxidase techniques. A number of antibodies were identified wliich reacted with similar populations of lymphocytes from vervet monkeys. These included antibodies that define T cells, suppressor/cytotoxic cell subset, pan B cells, monocytes and MHC class 1 and II. A number of anti-CD4 markers examined were unsuitable for use in the vervet system. Dr Joseph O. Olobo, Institute of Primate Research, National Museums of Kenya, P.O. Box 24481, Karen. Nairobi. Kenya
Determination of lymphocyte subsets is showing increasing application in the definition of host immunologicai status in association with infectious disease agents [1]. Such application also extends into the areas of immunodeficiency syndromes and other pathological conditions [1]. The advent of monoclonal antibodies in combination with eitherflowcytometry or immunoperoxidase techniques has greatly simplified enumeration of lymphocyte subsets. Recent work has demonstrated that certain monoclonal antibodies against human lymphocyte antigens are also suitable for differentiation of lymphocyte subsets of non-human primates [2, 3]. Current studies in this laboratory concern the utilization of an established vervet monkey/ Leishmania major disease model [4] to examine host immunologicai responses following vaccination with candidate vaccine preparations. Of importance in this respect is the screening of available anti-human lymphocyte markers for their cross-reactivity with vervet monkey lymphocytes. This report summarizes the results of a study investigating this aspect. MATERIALS AND METHODS Animals, Adult vervet monkeys {Cercopithecus aethiops). free from overt disease as ascertained during a 3-
month quarantine period at the Animal Facility, Institute of Primate Research, were used for the investigation. Animal maintenance and care and anaesthetic procedures for bleedings have been described elsewhere 15]. Isolation of lymphocytes and specimen preparation. Peripheral blood mononuclear cells (PBMC) were isolated over Ficoll-Hypaque from venous blood diluted in an equal volume of Alsever's solution [5]. For immunoperoxidase staining, purified PBMC were adjusted at 2xlO'/ml of phosphate-buflered saline (PBS) and cytospin smears were prepared on microscope slides using a Cytospin 2 (Shandon Scientific Ltd, UK), air dried and fixed in acetone before staining. Immunoperoxidase staining. An indirect method was used for staining fixed cytocentrifuge cell smears [6, 7]. The primary antibodies used are listed in Table I. Biotin-labelled affinity pure goat anti-mouse Ig (Sigma, Poole, UK) diluted 15-fold served as the second antibody followed by ExtrAvidin^" peroxidase (Sigma) diluted 1:15 in modified PBS. Colour reaction product was developed with the chromogen 3-amino-9-ethyIcarbazole (AEC, Sigma) [6]. Counterstaining was performed with haematoxylin-2 (Sigma). The slides were mounted using Tris-glycerol buffer. A total of 200 cells were examined to determine the percentage of positive ones. Immunofluoreseence staining of lymphocytes and FACScan analysis. Two staining procedures were used depending on the source of monoclonal antibody. Wliole blood staining procedure was performed according to the instructions supplied (Ortho-mune Diagnostic Systems, USA). Ficoll-Paque purified lymphocytes were stained by either direct or indirect immunofluoreseence techniques
199
200
/ . O. Olobo TABLE I. Antibodies used for staining peripheral blood leucocytes from vervet monkeys Antibody
CD group
Til
CD2
UCHTl FNI8 GM12 OKT4 Leu 3a QS4I20 66.1 101-69 AI/64 HP26 Leul Leu2a UCHT4 T8 UCHMI 2D1 UCHLI Leu7 Leull B9.I2.1 (Class I) B7.21.2.DP (Class II) D1.I2.DR BT3/4DQ 2B2M
group CD3 CD3 (B cells) CD4 CD4 CD4 CD4 CD4 CD4 CD4 CD5 CD8 CD8 CD8 CDI4 CD45 CD45RO CD 57 CD16 — — — — —
Reactivity
Source
Coulter, France P. C. L. Beverley, ICRF, London, UK M. Jonker, TNO Primate Center, The Netherlands M. Jonker, TNO Primate Center, The Netherlands Ortho-mune, USA Becton-Dickinson, USA P. C. L. Beverley, ICRF, London, UK S. Carrel, LICR, Switzerland S. Carrel, LICR, Switzerland S. Carrel, LICR, Switzerland S. Carrel, LICR, Switzerland Becton-Dickinson, USA Becton-Dickinson, USA P. C. L. Beverley, ICRF, London, UK Dakopatts, Denmark P. C. L. Beverley, ICRF, London, UK P. C. L. Beverley, ICRF, London, UK P. C. L. Beverley, ICRF, London, UK Becton-Dickinson, USA Becton-Dickinson, USA S. Carrel, LICR, Switzerland S. Carrel, LICR, Switzerland S. Carrel, LICR, Switzerland S. Carrel, LICR, Switzerland S. Carrel, LICR, Switzerland
+ + + = strongly positive; -I- + = positive; -t- = weakly positive; — = negative. Whole blood or purified PBMC were stained by either direct or indirect immunofluoreseence methods and 2500 lymphocytes were analysed by flow cytometry. An indirect immunoperoxidase staining procedure was used and 200 cells were counted before determining the percentage of positive cells. [8] employing antibodies listed in Table I. FITCconjugated sheep anti-mouse Ig (Amersham, UK), diluted 50-fold in immunofluoreseence buffer [8], was the secondary reagent. Stained cells were fixed for at least I h in 2% formalin in PBS before analysis. A FACScan flow cytometer (Becton-Dickinson, Mountain View, CA, USA) was used for cell analysis. A total of 2500 viable cells from each sample were run before computing the percentage of positive cells. Stained cells were analysed by both forward and right-angle light scattering properties [9].
RESULTS Table I shows the degree of reactivity of the antibodies with vervet monkey peripheral blood mononuclear cells. Strong reactivities were obtained with monoclonal antibodies that define T cells, suppressor/cytotoxic cell subset, pan B cells, monocytes and MHC class I and II. Of interest was the finding that none of the CD4 markers tested was found to identify a similar population of cells in vervet monkeys (Table I).
DISCUSSION This study shows that a number of anti-human lymphocyte markers are also suitable for identification of lymphocyte subsets in the vervet monkey. Similar studies conducted using some of these antibodies in other species of non-human primates gave comparable results [3, 10, 11]. The presence of similar antigens in both human and vervet monkeys and non-human primate species from other parts ofthe world indicates that these epitopes were conserved during evolution. Of interest was the finding that of all the antihuman CD4 markers used, none recognized a similar lymphocyte subpopulation in the vervet monkey. This implies that either there are too few CD4 cells in vervet monkey PBMC or they express a different epitope not recognized by any of the antibodies used. Similar results had been reported earlier [3] using monoclonal antibody OKT4 on vervet monkey peripheral blood lymphocytes.
Vervet Monkey Lymphocyte Phenotypes When immunoperoxidase assays were compared with flow cytometry analysis, a correlation was found between the two procedures. However, the former was cheaper but more laborious as compared with the latter. Availability ofa battery of markers for vervet monkey lymphocytes will support the current leishmania vaccine development studies and should serve as a reference list for investigators involved in biomedical studies utilizing this nonhuman primate species.
ACKNOWLEDGMENTS This study was supported in part by the UNDP/ World Bank/WHO Special Programme for Research and Training in Tropical Diseases and the Rockefeller Foundation.
REFERENCES 1 Westermann J, Pabst R. Lymphocyte subsets in the blood: A diagnostic window on the lymphoid system? Immunol Today 1990;ll:406-10. 2 Ahmed-Ansari A, Brodie AR, Fultz PN, Anderson DC, Sell KW, McClure HM. Flow microfluorometric analysis of peripheral blood mononuclear cells from nonhuman primates: Correlation of phenotype with immune function. Am J Primatol 1989; 17:107-31.
201
3 Martin LN, Gormus BJ, Bozelka BE. Functional analysis of monkey lymphocyte subsets defined by 0KT4 and OKT8 monoclonal antibodies. Cell Immunol 1983;77:338-47. 4 Githure JI, Reid GDF, Binhazim AA, Anjili CO, Shatry AM, Hendricks LD. Leishmania major: The suitability of East African nonhuman primates as animal models for cutaneous leishmaniasis. Exp Parasit 1987;64:438-47. 5 Olobo JO, Reid GDF. Mitogenie responses of peripheral blood mononuclear cells of vervet monkeys {Cercopiiliecus aethiops): Apparent role of adherent cells. Amer J Primatol 1990;20:31-6. 6 Paradis IL, Merrall EJ, Krell JM, Dauber JH, Rogers RM, Robin BS. Lymphocyte enumeration: A comparison between a modified avidin-biotinimmunoperoxidase system and fiow cytometry. J Histochem Cytochem 1984;32:358-62. 7 Ll CY, Ziesmer SC, Lazcano-Villareal L. Use of azide and hydrogen peroxide as an inhibitor for endogenous peroxidase in the immunoperoxidase method. J Histochem Cytochem 1987; 12:1457-60. 8 Olobo JO, Black SJ. Selected phenotypic and cloning properties of a bovine lymphoblastoid cell line, BL20. Vet Immunol Immunopath 1989;20:165-72. 9 Herzenberg LA, Herzenberg LA. Mouse immunoglobulin allotypes: Description and special methodology. In: Weir DM, ed. Handbook of Experimental Immunology. Oxford: Blackwell Scientific Publications, 1978:12.1-23. 10 Neubauer RJ, Briggs CJ, Noer KB, Robin H. Identification of normal and transformed lymphocyte subsets of nonhuman primates with monoclonal antibodies to human lymphocytes. J Immunol I983;l30:1323-9. 11 Letvin NL, Kin NW, Reinherz EL, Hunt RD, Lane JH, Schlossman SF. T lymphocyte surface antigens in primates. Eur J Immunol 1983; 13:345-7.
Reactivity of some Monoclonal Antibodies Specific for Human Lymphocytes with Vervet Monkey Peripheral Blood Mononuclear Cells J. O. OLOBO Institute of Primate Research, National Museums of Kenya, Nairobi, Kenya
Olobo JO. Reactivity of some Monoclonal Antibodies Specific for Human Lymphocytes with Vervet Monkey Peripheral Blood Mononuclear Cells. Scand J Immunol 1992;36(Suppl. 11):I99201 Reactivities of some monoclonal antibodies to human lymphocyte surface antigens were tested on vervet monkey peripheral blood mononuclear cells (PBMC), using flow cytometry and immunoperoxidase techniques. A number of antibodies were identified wliich reacted with similar populations of lymphocytes from vervet monkeys. These included antibodies that define T cells, suppressor/cytotoxic cell subset, pan B cells, monocytes and MHC class 1 and II. A number of anti-CD4 markers examined were unsuitable for use in the vervet system. Dr Joseph O. Olobo, Institute of Primate Research, National Museums of Kenya, P.O. Box 24481, Karen. Nairobi. Kenya
Determination of lymphocyte subsets is showing increasing application in the definition of host immunologicai status in association with infectious disease agents [1]. Such application also extends into the areas of immunodeficiency syndromes and other pathological conditions [1]. The advent of monoclonal antibodies in combination with eitherflowcytometry or immunoperoxidase techniques has greatly simplified enumeration of lymphocyte subsets. Recent work has demonstrated that certain monoclonal antibodies against human lymphocyte antigens are also suitable for differentiation of lymphocyte subsets of non-human primates [2, 3]. Current studies in this laboratory concern the utilization of an established vervet monkey/ Leishmania major disease model [4] to examine host immunologicai responses following vaccination with candidate vaccine preparations. Of importance in this respect is the screening of available anti-human lymphocyte markers for their cross-reactivity with vervet monkey lymphocytes. This report summarizes the results of a study investigating this aspect. MATERIALS AND METHODS Animals, Adult vervet monkeys {Cercopithecus aethiops). free from overt disease as ascertained during a 3-
month quarantine period at the Animal Facility, Institute of Primate Research, were used for the investigation. Animal maintenance and care and anaesthetic procedures for bleedings have been described elsewhere 15]. Isolation of lymphocytes and specimen preparation. Peripheral blood mononuclear cells (PBMC) were isolated over Ficoll-Hypaque from venous blood diluted in an equal volume of Alsever's solution [5]. For immunoperoxidase staining, purified PBMC were adjusted at 2xlO'/ml of phosphate-buflered saline (PBS) and cytospin smears were prepared on microscope slides using a Cytospin 2 (Shandon Scientific Ltd, UK), air dried and fixed in acetone before staining. Immunoperoxidase staining. An indirect method was used for staining fixed cytocentrifuge cell smears [6, 7]. The primary antibodies used are listed in Table I. Biotin-labelled affinity pure goat anti-mouse Ig (Sigma, Poole, UK) diluted 15-fold served as the second antibody followed by ExtrAvidin^" peroxidase (Sigma) diluted 1:15 in modified PBS. Colour reaction product was developed with the chromogen 3-amino-9-ethyIcarbazole (AEC, Sigma) [6]. Counterstaining was performed with haematoxylin-2 (Sigma). The slides were mounted using Tris-glycerol buffer. A total of 200 cells were examined to determine the percentage of positive ones. Immunofluoreseence staining of lymphocytes and FACScan analysis. Two staining procedures were used depending on the source of monoclonal antibody. Wliole blood staining procedure was performed according to the instructions supplied (Ortho-mune Diagnostic Systems, USA). Ficoll-Paque purified lymphocytes were stained by either direct or indirect immunofluoreseence techniques
199
200
/ . O. Olobo TABLE I. Antibodies used for staining peripheral blood leucocytes from vervet monkeys Antibody
CD group
Til
CD2
UCHTl FNI8 GM12 OKT4 Leu 3a QS4I20 66.1 101-69 AI/64 HP26 Leul Leu2a UCHT4 T8 UCHMI 2D1 UCHLI Leu7 Leull B9.I2.1 (Class I) B7.21.2.DP (Class II) D1.I2.DR BT3/4DQ 2B2M
group CD3 CD3 (B cells) CD4 CD4 CD4 CD4 CD4 CD4 CD4 CD5 CD8 CD8 CD8 CDI4 CD45 CD45RO CD 57 CD16 — — — — —
Reactivity
Source
Coulter, France P. C. L. Beverley, ICRF, London, UK M. Jonker, TNO Primate Center, The Netherlands M. Jonker, TNO Primate Center, The Netherlands Ortho-mune, USA Becton-Dickinson, USA P. C. L. Beverley, ICRF, London, UK S. Carrel, LICR, Switzerland S. Carrel, LICR, Switzerland S. Carrel, LICR, Switzerland S. Carrel, LICR, Switzerland Becton-Dickinson, USA Becton-Dickinson, USA P. C. L. Beverley, ICRF, London, UK Dakopatts, Denmark P. C. L. Beverley, ICRF, London, UK P. C. L. Beverley, ICRF, London, UK P. C. L. Beverley, ICRF, London, UK Becton-Dickinson, USA Becton-Dickinson, USA S. Carrel, LICR, Switzerland S. Carrel, LICR, Switzerland S. Carrel, LICR, Switzerland S. Carrel, LICR, Switzerland S. Carrel, LICR, Switzerland
+ + + = strongly positive; -I- + = positive; -t- = weakly positive; — = negative. Whole blood or purified PBMC were stained by either direct or indirect immunofluoreseence methods and 2500 lymphocytes were analysed by flow cytometry. An indirect immunoperoxidase staining procedure was used and 200 cells were counted before determining the percentage of positive cells. [8] employing antibodies listed in Table I. FITCconjugated sheep anti-mouse Ig (Amersham, UK), diluted 50-fold in immunofluoreseence buffer [8], was the secondary reagent. Stained cells were fixed for at least I h in 2% formalin in PBS before analysis. A FACScan flow cytometer (Becton-Dickinson, Mountain View, CA, USA) was used for cell analysis. A total of 2500 viable cells from each sample were run before computing the percentage of positive cells. Stained cells were analysed by both forward and right-angle light scattering properties [9].
RESULTS Table I shows the degree of reactivity of the antibodies with vervet monkey peripheral blood mononuclear cells. Strong reactivities were obtained with monoclonal antibodies that define T cells, suppressor/cytotoxic cell subset, pan B cells, monocytes and MHC class I and II. Of interest was the finding that none of the CD4 markers tested was found to identify a similar population of cells in vervet monkeys (Table I).
DISCUSSION This study shows that a number of anti-human lymphocyte markers are also suitable for identification of lymphocyte subsets in the vervet monkey. Similar studies conducted using some of these antibodies in other species of non-human primates gave comparable results [3, 10, 11]. The presence of similar antigens in both human and vervet monkeys and non-human primate species from other parts ofthe world indicates that these epitopes were conserved during evolution. Of interest was the finding that of all the antihuman CD4 markers used, none recognized a similar lymphocyte subpopulation in the vervet monkey. This implies that either there are too few CD4 cells in vervet monkey PBMC or they express a different epitope not recognized by any of the antibodies used. Similar results had been reported earlier [3] using monoclonal antibody OKT4 on vervet monkey peripheral blood lymphocytes.
Vervet Monkey Lymphocyte Phenotypes When immunoperoxidase assays were compared with flow cytometry analysis, a correlation was found between the two procedures. However, the former was cheaper but more laborious as compared with the latter. Availability ofa battery of markers for vervet monkey lymphocytes will support the current leishmania vaccine development studies and should serve as a reference list for investigators involved in biomedical studies utilizing this nonhuman primate species.
ACKNOWLEDGMENTS This study was supported in part by the UNDP/ World Bank/WHO Special Programme for Research and Training in Tropical Diseases and the Rockefeller Foundation.
REFERENCES 1 Westermann J, Pabst R. Lymphocyte subsets in the blood: A diagnostic window on the lymphoid system? Immunol Today 1990;ll:406-10. 2 Ahmed-Ansari A, Brodie AR, Fultz PN, Anderson DC, Sell KW, McClure HM. Flow microfluorometric analysis of peripheral blood mononuclear cells from nonhuman primates: Correlation of phenotype with immune function. Am J Primatol 1989; 17:107-31.
201
3 Martin LN, Gormus BJ, Bozelka BE. Functional analysis of monkey lymphocyte subsets defined by 0KT4 and OKT8 monoclonal antibodies. Cell Immunol 1983;77:338-47. 4 Githure JI, Reid GDF, Binhazim AA, Anjili CO, Shatry AM, Hendricks LD. Leishmania major: The suitability of East African nonhuman primates as animal models for cutaneous leishmaniasis. Exp Parasit 1987;64:438-47. 5 Olobo JO, Reid GDF. Mitogenie responses of peripheral blood mononuclear cells of vervet monkeys {Cercopiiliecus aethiops): Apparent role of adherent cells. Amer J Primatol 1990;20:31-6. 6 Paradis IL, Merrall EJ, Krell JM, Dauber JH, Rogers RM, Robin BS. Lymphocyte enumeration: A comparison between a modified avidin-biotinimmunoperoxidase system and fiow cytometry. J Histochem Cytochem 1984;32:358-62. 7 Ll CY, Ziesmer SC, Lazcano-Villareal L. Use of azide and hydrogen peroxide as an inhibitor for endogenous peroxidase in the immunoperoxidase method. J Histochem Cytochem 1987; 12:1457-60. 8 Olobo JO, Black SJ. Selected phenotypic and cloning properties of a bovine lymphoblastoid cell line, BL20. Vet Immunol Immunopath 1989;20:165-72. 9 Herzenberg LA, Herzenberg LA. Mouse immunoglobulin allotypes: Description and special methodology. In: Weir DM, ed. Handbook of Experimental Immunology. Oxford: Blackwell Scientific Publications, 1978:12.1-23. 10 Neubauer RJ, Briggs CJ, Noer KB, Robin H. Identification of normal and transformed lymphocyte subsets of nonhuman primates with monoclonal antibodies to human lymphocytes. J Immunol I983;l30:1323-9. 11 Letvin NL, Kin NW, Reinherz EL, Hunt RD, Lane JH, Schlossman SF. T lymphocyte surface antigens in primates. Eur J Immunol 1983; 13:345-7.
