Comparative analysis of CD4-mediated down-regulation of T cell adhesion to B cells by flow cytometry and fluorescence microscopy.
We have previously shown by means of fluorescence microscopy that antigen-independent adhesion of resting CD4 T cells to EBV-transformed B cells can be down-regulated by ligand interaction with CD4. In this study we used flow cytometry analysis of conjugate formation to confirm these findings. No conjugates between resting CD4 + T cells and B cells were initially detected in the latter method, because flow velocity in the flow chamber induces hydrodynamic elongation forces which disrupt low-affinity conjugates. After forcing cell conjugation by low-speed centrifugation of T and B cells, conjugates became detectable although in smaller numbers than in fluorescence microscopy. "Forced" cell conjugates had similar characteristics to their unforced counterparts, i.e., 37 degrees C temperature dependency, mediation by LFA-1/ICAM-1 and CD2/LFA-3 pathways, and transiency. The latter characteristic was at least partly mediated by CD4/HLA class II interaction, since adhesion of CD4 + T cells to HLA class II-B cells was more stable. In addition, adhesion was inhibited by anti-CD4 antibodies but not by an HLA DR-derived peptide known to inhibit unforced CD4 + T cell adhesion to B cells. This blocking effect was partially reproduced by reducing the centrifugation time prior to the adhesion assay. These results show that a) CD4-mediated down-regulation of T cell adhesion can be observed by means of two different techniques, and b) analysis of cell-cell adhesion after increasing centrifugation times (and possibly speeds) is a simple way of measuring adhesion forces semiquantitatively.
Hauss
P
P
INSERM U429, Hôpital Necker-Enfants Malades, Paris, France.
Selz
F
F
Fischer
A
A
D000328:Adult; D000906:Antibodies / Q000494:pharmacology; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology*; D018801:CD2 Antigens / Q000276:immunology; D015704:CD4 Antigens / Q000276:immunology*; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D002448:Cell Adhesion / Q000276:immunology*; D002478:Cells, Cultured; D002498:Centrifugation / Q000379:methods; D015536:Down-Regulation / Q000276:immunology*; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods; D000949:Histocompatibility Antigens Class II / Q000276:immunology; D006801:Humans; D016169:Lymphocyte Function-Associated Antigen-1 / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D013696:Temperature; D013997:Time Factors
D000906:Antibodies; D018801:CD2 Antigens; D015704:CD4 Antigens; D000949:Histocompatibility Antigens Class II; D016169:Lymphocyte Function-Associated Antigen-1