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Contribution of three-color cytofluorimetry to the kinetic analysis of the switch from CD45RA to CD45RO: Study of the in vitro activated CD8+ T lymphocytes model
261 FLOW CYTOMETRIC MONITORING OF IMMUNOMODULATORS IN MOUSE MODEL. CARAYON Pierre, PETITPRETRE G~raldine, BORD Annie, COUSIN Xavier, and CASELLAS Pierre, Sanofi
Recherche, Montpe/lier, FRANCE. An immunopharmacological model using flow cytometry has been developed to study the effects of drugs on the activation of lymphocytes in vivo. T lymphocytes were activated following administration into BALB/c mice of 25 pg anti-mouse CD3 antibody via the lateral tail vein. T-cell activation was evaluated 24 hours later by measuring the expression of intedeukin-2 receptors, transferrin receptors and the size of Thy 1.2-positive splenic T lymphocyles. B lymphocyte activation was monitored by the upregulation of MHC-class II antigen (I-Ad). Moreover, anti-CD3 antibody induced an infiltration of polymorphonuclear cells (PMN) into the spleen. The activation of this leukocyte subset can be evaluated by measuring the increased expression of the CD11b (Mac-1) antigen. Effects of Cyclosporin-A (CsA) and Dexamethasone (DEX) were studied in this model. When administered by i.p. route, CsA strongly inhibited T-cell activation. 50% inhibition was observed at the dose of 1 mg/kg. However, CsA had a poor effect on B-cell activation and no effect on PMN infiltration. By contrast, DEX was poorly efficient in this model, doses higher than 300 mg/kg being necessary to inhibit T-cell activation.
QUANTIFICATION OF C D I l a , ctCIIAIN OF LFA-I M O L E C U L E , ON DIFFERENT LYMPIIOCYTE SUBPOPULATIONS. PORTALES Pierre., BOURDIOL Luc. and CLOT Jacques. Laboratoire d7mmtmologie, INSERM U291, Hrpital Saint-Ehfi CHR Montpel/ier, France. Integrins contains u :rod 13subunits which are non covalcntly associated. The integrin 112 family consists in three molecules (LFA- I, Mac1 and p 150, 95). The LFA- 1 (Leukocyte Function Associated- 1) molecule is expressed on lymphocytes and its ligand on the target cells is ICAM-I or 1CAM-2 (Intercellul:tr Cell Adhesion Molecules). The aim of this study wits to ev,'duate the density of LFA+ 1 molecules on different lymphocyte populations (CD3 +, CD4 +, CD8 + and CDI9+), by studying tile (:t oh:tin (CDI la), specific of the molecule. Material and method: Fresh peripheral whole blood from 10 heuhhy aduhs wits collected on EDTA. Lymphocytes ,,,,,eredouble labeled by a direct inununofluorescence technic using a CDI la monocloual antibody (MAb) conjugated with fluorescei'ne isothit~yanale (FITC) anti :t second MAb to CD3, CD4, CD8 or CDI9 conjugated with phycocrythrine (PE), all from llnmunotech (France). A selective acquisition of the different subpopulations oil a flow cylometer (FACScan, Beckton Dickinson) was performed, and tile FITC-CD 1la Iluorescence intensity was analysed. The median of fluorescence intensity (M FI) of CDI la molectdes on the different populations of each subject was compared to a standart curve established with a standart quantification kit (Beckton Dickinson) expressing (1, 280{), 9300, 18000 or 3300{) molecules of equivalent soluble fluorochrome (MESF). Resuhs : Tile CDI llt density was clearly different according to the lymphocyte subset. CD4 + T cells carried 3594 + 646 (nlean + SEM) CDI la molecules per cell, whereas CD8 + cells expressedf6867 + 1575 distributed into two populations of different density (2927 + 401 and 8641 + 1056). Lastly, CDI9+ cells only brought 1167 + 167 CDI la molecules per cell. Discussion : These data pointed out tile heterogeneity of LFA- I molecule expression on lymphocytes.
50a
C o n t r i b u t i o n o f t h r e e - c o l o r c y t o f l u o r i m e t r y to the kinetic analysis of the switch from CD45RA to C D 4 5 R O : study of the in vitro activated C D 8 + T lymphocytes model. Leila BENTOUATI, Jean TKA(~ZI.IK, Philippe M()INARD, Michel ABBAL, Elie OHAYON. Laboratoire dTmmunologie - CHU Rangueil - 31054 TOULOUSE C~dex The CD45 family is constituted by different membrane glycoproteins deriving from a single gene by alternative splicing. During lymphocyte activation, switch from CD45RA to CD45RO isoform occurs leading to transformation of naive cells into memory cells. Among the cytotoxic/suppressor CD8 + T lymphocytes of normal adults, the CD45RA (naive) molecule is predominant, while CD45RO (memory) is the most frequent isoform among CD4 + cells. Since CD8+CD45 switch is supposed to be implicated during graft rejection, we tried to analyze this phenomenon among CD8 + cells in vin'o, in order to develop the most appropriate strategy for dating it. Peripheral blood lymphocytes from normal donors were separated and bulk cultured for 10 days in RPMI supplemented with 10%SVF, 10 U/ml of r-lL2 and stimulated with either PHA, or anti-CD3, or anti-CD3+anti-CD28...Cells were analyzed every day on a FACScan (BD) by a three-color labeling technique using : anti-CDS-PERCP (BD), anti-CD45RA-FITC (BD) and anti-CD45RO-PE (DAKO). Raw data were analyzed through LYSYS II software, using different gating procedures (regions, inter-regions) and data expression (histograms, dot-plots). This study allowed the establishment of kinetic events : CDS+CD45RO + increase, CD8+CD45RA + decrease among total lymphocytes and CD45RA+CD45RO + double-positive cells evolution among CD8 + . Our aim to stress interindividual differences, showed that the considered criterias arc not of the same value : 1/No interindividual difference is observed when the switching time is defined either by the time of intersection between the CD8+CD45RA + and CD8+CD45RO + curves, or by the peak of CD8+CD45RA+Iow CD45RO + low triple-positive cells. In both cases the event occurs at day 3 for all donors tested. 2/Interindividual differences are objectived when one is looking at either the slope of CD8+CD45RO + curve or the time lasting untill the percentage of CD8+CD45RO ÷ cells reaches its "plateau value". In conclusion : the occurence of triple positive cells at day 3 could be a good tool for dating initiation of rejection phenomenon in grafted patients ; even if we can assume that the in vivo kinetic events in presence of immunosuppressive drug are probably different.
FLOW CYTOMETRIC DETECTION OF INTRACELLULAR EXPRESSION OF CYTOKINES AFTER ACTIVATION OF PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC) IN VITRO. OVIGNE Jean-Marc, BORD Annie, CASELLAS Pierre, PETITPRETKE G&aldine, and CARAYON Pierre. The intracellular detection of five different cytokines (IFN-gamma, TNFalpha, IL-I beta, IL-2, and IL-6) was carried out following activation of PBMC by PMA + ionomycin. The paraformaldehyde-saponin procedure was used as described (1). IFN-gamma, TNF-alpha and IL-2 were detected within 10 hours following the activation. Intracellular IL-1 beta and IL-6 were not detectable using this type of activation. This procedure allows the characterization of cytokine-producing cells by combining intracytoplasmic immunofluorescence and surface staining. However, two major problems need to be solved to improve this technique. Firstly, the problem of sensitivity which is not high enough to detect cells producing low levels of cytokines. Secondly, because the cytokines can be detected at the cell surface as well, this enables the labelling of cells bearing cytokines at their surface via cytokine receptors. (I) SANDER B., ANDERSON J., and ANDERSON U. (1991) Immunological Reviews, 119: 65-93.
Recherche, Montpe/lier, FRANCE. An immunopharmacological model using flow cytometry has been developed to study the effects of drugs on the activation of lymphocytes in vivo. T lymphocytes were activated following administration into BALB/c mice of 25 pg anti-mouse CD3 antibody via the lateral tail vein. T-cell activation was evaluated 24 hours later by measuring the expression of intedeukin-2 receptors, transferrin receptors and the size of Thy 1.2-positive splenic T lymphocyles. B lymphocyte activation was monitored by the upregulation of MHC-class II antigen (I-Ad). Moreover, anti-CD3 antibody induced an infiltration of polymorphonuclear cells (PMN) into the spleen. The activation of this leukocyte subset can be evaluated by measuring the increased expression of the CD11b (Mac-1) antigen. Effects of Cyclosporin-A (CsA) and Dexamethasone (DEX) were studied in this model. When administered by i.p. route, CsA strongly inhibited T-cell activation. 50% inhibition was observed at the dose of 1 mg/kg. However, CsA had a poor effect on B-cell activation and no effect on PMN infiltration. By contrast, DEX was poorly efficient in this model, doses higher than 300 mg/kg being necessary to inhibit T-cell activation.
QUANTIFICATION OF C D I l a , ctCIIAIN OF LFA-I M O L E C U L E , ON DIFFERENT LYMPIIOCYTE SUBPOPULATIONS. PORTALES Pierre., BOURDIOL Luc. and CLOT Jacques. Laboratoire d7mmtmologie, INSERM U291, Hrpital Saint-Ehfi CHR Montpel/ier, France. Integrins contains u :rod 13subunits which are non covalcntly associated. The integrin 112 family consists in three molecules (LFA- I, Mac1 and p 150, 95). The LFA- 1 (Leukocyte Function Associated- 1) molecule is expressed on lymphocytes and its ligand on the target cells is ICAM-I or 1CAM-2 (Intercellul:tr Cell Adhesion Molecules). The aim of this study wits to ev,'duate the density of LFA+ 1 molecules on different lymphocyte populations (CD3 +, CD4 +, CD8 + and CDI9+), by studying tile (:t oh:tin (CDI la), specific of the molecule. Material and method: Fresh peripheral whole blood from 10 heuhhy aduhs wits collected on EDTA. Lymphocytes ,,,,,eredouble labeled by a direct inununofluorescence technic using a CDI la monocloual antibody (MAb) conjugated with fluorescei'ne isothit~yanale (FITC) anti :t second MAb to CD3, CD4, CD8 or CDI9 conjugated with phycocrythrine (PE), all from llnmunotech (France). A selective acquisition of the different subpopulations oil a flow cylometer (FACScan, Beckton Dickinson) was performed, and tile FITC-CD 1la Iluorescence intensity was analysed. The median of fluorescence intensity (M FI) of CDI la molectdes on the different populations of each subject was compared to a standart curve established with a standart quantification kit (Beckton Dickinson) expressing (1, 280{), 9300, 18000 or 3300{) molecules of equivalent soluble fluorochrome (MESF). Resuhs : Tile CDI llt density was clearly different according to the lymphocyte subset. CD4 + T cells carried 3594 + 646 (nlean + SEM) CDI la molecules per cell, whereas CD8 + cells expressedf6867 + 1575 distributed into two populations of different density (2927 + 401 and 8641 + 1056). Lastly, CDI9+ cells only brought 1167 + 167 CDI la molecules per cell. Discussion : These data pointed out tile heterogeneity of LFA- I molecule expression on lymphocytes.
50a
C o n t r i b u t i o n o f t h r e e - c o l o r c y t o f l u o r i m e t r y to the kinetic analysis of the switch from CD45RA to C D 4 5 R O : study of the in vitro activated C D 8 + T lymphocytes model. Leila BENTOUATI, Jean TKA(~ZI.IK, Philippe M()INARD, Michel ABBAL, Elie OHAYON. Laboratoire dTmmunologie - CHU Rangueil - 31054 TOULOUSE C~dex The CD45 family is constituted by different membrane glycoproteins deriving from a single gene by alternative splicing. During lymphocyte activation, switch from CD45RA to CD45RO isoform occurs leading to transformation of naive cells into memory cells. Among the cytotoxic/suppressor CD8 + T lymphocytes of normal adults, the CD45RA (naive) molecule is predominant, while CD45RO (memory) is the most frequent isoform among CD4 + cells. Since CD8+CD45 switch is supposed to be implicated during graft rejection, we tried to analyze this phenomenon among CD8 + cells in vin'o, in order to develop the most appropriate strategy for dating it. Peripheral blood lymphocytes from normal donors were separated and bulk cultured for 10 days in RPMI supplemented with 10%SVF, 10 U/ml of r-lL2 and stimulated with either PHA, or anti-CD3, or anti-CD3+anti-CD28...Cells were analyzed every day on a FACScan (BD) by a three-color labeling technique using : anti-CDS-PERCP (BD), anti-CD45RA-FITC (BD) and anti-CD45RO-PE (DAKO). Raw data were analyzed through LYSYS II software, using different gating procedures (regions, inter-regions) and data expression (histograms, dot-plots). This study allowed the establishment of kinetic events : CDS+CD45RO + increase, CD8+CD45RA + decrease among total lymphocytes and CD45RA+CD45RO + double-positive cells evolution among CD8 + . Our aim to stress interindividual differences, showed that the considered criterias arc not of the same value : 1/No interindividual difference is observed when the switching time is defined either by the time of intersection between the CD8+CD45RA + and CD8+CD45RO + curves, or by the peak of CD8+CD45RA+Iow CD45RO + low triple-positive cells. In both cases the event occurs at day 3 for all donors tested. 2/Interindividual differences are objectived when one is looking at either the slope of CD8+CD45RO + curve or the time lasting untill the percentage of CD8+CD45RO ÷ cells reaches its "plateau value". In conclusion : the occurence of triple positive cells at day 3 could be a good tool for dating initiation of rejection phenomenon in grafted patients ; even if we can assume that the in vivo kinetic events in presence of immunosuppressive drug are probably different.
FLOW CYTOMETRIC DETECTION OF INTRACELLULAR EXPRESSION OF CYTOKINES AFTER ACTIVATION OF PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC) IN VITRO. OVIGNE Jean-Marc, BORD Annie, CASELLAS Pierre, PETITPRETKE G&aldine, and CARAYON Pierre. The intracellular detection of five different cytokines (IFN-gamma, TNFalpha, IL-I beta, IL-2, and IL-6) was carried out following activation of PBMC by PMA + ionomycin. The paraformaldehyde-saponin procedure was used as described (1). IFN-gamma, TNF-alpha and IL-2 were detected within 10 hours following the activation. Intracellular IL-1 beta and IL-6 were not detectable using this type of activation. This procedure allows the characterization of cytokine-producing cells by combining intracytoplasmic immunofluorescence and surface staining. However, two major problems need to be solved to improve this technique. Firstly, the problem of sensitivity which is not high enough to detect cells producing low levels of cytokines. Secondly, because the cytokines can be detected at the cell surface as well, this enables the labelling of cells bearing cytokines at their surface via cytokine receptors. (I) SANDER B., ANDERSON J., and ANDERSON U. (1991) Immunological Reviews, 119: 65-93.