Clustering of dendritic cells, helper T lymphocytes, and histocompatible B cells during primary antibody responses in vitro.
Mouse spleen suspensions generate discrete cell clusters within 1-2 d of culture. We have isolated these clusters by velocity sedimentation to study their contribution to primary antibody responses. Clusters represent approximately 5% of the starting spleen cells and consist of 20-50% B cells, 20-50% T cells, and 10-20% dendritic cells (DC). When the cultures are stimulated with thymus-dependent antigens, like heterologous red cells or dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH), the clusters are the principal site for the development of plaque-forming cells (PFC). Noncluster fractions form few PFC and only when supplemented with fresh DC. PFC responses in all cases are antigen specific. B cells cluster only in the presence of T cells and DC (1 DC/200 B-T cell mixtures) and only after encountering specific antigen. The elimination of either DC or Lyt-1+2- T cells, with monoclonal antibody and complement, ablates B cell development into PFC. PFC responses are restored with antigen-nonspecific helper factors formed in the syngeneic mixed leukocyte reaction between DC and T cells. Since PFC to DNP-KLH do not develop de novo when B cells are exposed to antigen and helper factors, anti-DNP PFC precursors must be stimulated within clusters to become responsive to helper factors. PFC development within clusters is restricted by the major histocompatibility complex (MHC). When DC and T cells are from strain P1, then P1 but not P2 B cells develop into PFC; when DC are from strain P2 and T cells from strain P1, strain P2 B cells are selected to become PFC in clusters. The entry of B cells into clusters is itself MHC restricted, since P1 DC/T cells aggregate six times as many B cells from strain P1 as strain P2. Thus, clusters are the site in which DC, B, and T cells interact to generate PFC. One can use clusters to retrieve B cells that have been selected in an antigen-dependent, MHC-restricted fashion and to show that clustering B cells become responsive to soluble, polyclonal helper factors.
D000818:Animals; D000921:Antibody-Producing Cells / Q000378:metabolism; D001402:B-Lymphocytes / Q000276:immunology / Q000502:physiology*; D002449:Cell Aggregation; D000939:Epitopes; D005260:Female; D006133:Growth Substances / Q000502:physiology; D006183:H-2 Antigens / Q000235:genetics*; D006433:Hemocyanins / Q000276:immunology; D006462:Hemolytic Plaque Technique; D015847:Interleukin-4; D008211:Lymphocyte Cooperation; D008222:Lymphokines / Q000502:physiology; D008297:Male; D051379:Mice; D008805:Mice, Inbred A; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D012756:Sheep; D013154:Spleen / Q000166:cytology; D006377:T-Lymphocytes, Helper-Inducer / Q000276:immunology / Q000502:physiology*
D000939:Epitopes; D006133:Growth Substances; D006183:H-2 Antigens; D008222:Lymphokines; D015847:Interleukin-4; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin