Definition of conditions that enable antigen-specific activation of the majority of isolated trinitrophenol-binding B cells.

In an effort to further elucidate the early cellular events in generation of antibody responses, we have determined the requirements for antigen-specific initiation of the G0 to G1 transition by isolated trinitrophenol (TNP) -binding B lymphocytes. TNP-binding cells were isolated from normal B6D2F1 splenocyte populations using hapten affinity fractionation on disulfide-bonded TNP-gelatin-coated plates. Populations prepared in this way are greater than or equal to 96% immunoglobulin positive and 70-95% antigen binding. Isolated cells were cultured for 48 h in the presence of a variety of TNP conjugates including TNP-Brucella abortus (Ba), TNP-Ficoll, TNP-sheep erythrocytes (SRBC), TNP-human gamma globulin (HGG), or TNP-ovalbumin (OVA) before being harvested and subjected to acridine orange cell cycle analysis. As many as 80% of cells were in cycle by 48 h in response to TNP-Ba, a thymus-independent (TI1 antigen. A smaller proportion (congruent to 40%) were in cycle in response to TNP-Ficoll, a TI2 antigen. Significant activation was not detected in cultures challenged with the thymus-dependent immunogens TNP-SRBC, TNP-HGG, and TNP-OVA. Addition of interleukin 1 (IL-1), IL-2, B cell growth factor, and/or T cell-replacing factor to cultures did not facilitate responses to these immunogens, suggesting a requirement for antigen-specific T cell help for entry into cell cycle induced by thymus dependent antigens. Activation by TNP-Ba was antigen specific and independent of accessory cells, occurring with equal efficiency in bulk and single-cell cultures. Activation by TNP-Ba was inhibitable by anti-Fab and anti-mu antibodies, but not by anti-delta antibodies. Results indicate that activation of TNP-binding cells to enter cell cycle by TNP-Ba is independent of accessory cells and requires interaction of antigen with cell surface IgM. Exposure to thymus-dependent TNP-immunogens plus nonspecific helper factors is insufficient to cause entry of TNP-binding cells into cycle.

Isolated phosphorylcholine binding lymphocytes. I. Use of a cleavable crosslinking reagent for solid-phase adsorbent isolation of functional antigen binding cells.

Isolation and characterization of BALB/c lymphocytes specific for phosphorylcholine is described. The isolation protocol utilizes phosphorylcholine coupled to gelatin coated plates via the cleavable crosslinking reagent N-succinimidyl 3-(2-pyridyldiothio)propionate (SPDP). The procedure is rapid, requiring only 1-2 h and conducted entirely at 0-4 degrees C. Hapten binding cells are eluted by vigorous pipetting at 4 degrees C with medium containing 20% fetal calf serum. Approximately 70% of isolated cells rebind antigen as assessed using a PC Brucella abortus rosette assay while 50% express the TEPC15 idiotype. Approximately 85% of isolated and idiotype positive cells are B cells while the remainder are T cells. Limiting dilution analysis revealed that approximately 1/5 of PC binding cells isolated from the spleens of normal mice respond to lipopolysaccharide plus dextran sulfate by production of anti-PC antibody. Approximately 1/11 respond to PC Brucella abortus and 1/250 respond to PC sheep erythrocytes plus primed T cells by anti-PC antibody production. The results clearly demonstrate the effectiveness of this technique for isolation of highly enriched, functional, antigen specific lymphocytes.

Induction of clonally restricted thymus-dependent antibody responses in vitro using phosphorylcholine derivatized sheep erythrocytes.

Induction of anti-phosphorylcholine responses in vitro using phosphorylcholine derivatized sheep erythrocytes (SRBC) is described. These responses are thymus dependent and restricted to expression of the TEPC 15 idiotype. Optimal B-cell concentrations in cultures were shown to be different for elicitation of direct anti-PC PFC responses than for elicitation of PFC specific for carrier (SRBC) determinants. Similarly, these responses differed in optimal immunogen concentration requirements. These differences presumably relate to the clonality of the respective responses. The methodology described provides an alternative to induction of clonally restricted thymus-dependent responses in vitro using PC derivatized soluble protein antigens. Induction of responses using PC-SRBC has the advantages of the relative case of T-cell priming to SRBC and concurrent stimulation of monoclonal anti-PC responses and easily assayed polyclonal anti-SRBC responses.

Initiation of meiotic recombination by formation of DNA double-strand breaks: mechanism and regulation.

Homologous recombination is essential for accurate chromosome segregation during meiosis in most sexual organisms. Meiotic recombination is initiated by the formation of DSBs (DNA double-strand breaks) made by the Spo11 protein. We review here recent findings pertaining to protein-protein interactions important for DSB formation, the mechanism of an early step in the processing of Spo11-generated DSBs, and regulation of DSB formation by protein kinases.