Figure 3. B cells fail to receive contact-dependent help from SAP-deficient T cells.
a, Individual T-B contact durations (left) and their distribution (right). The non-cognate condition involves sap+/+ OT-2 cells non-specifically interacting with MD4 B cells following immunization with HEL-BSA mixed with OVA protein (137 measurements from 2 experiments). The cognate condition involves specific interactions between sap+/+ or sap−/− OT-2 T cells and MD4 B cells following immunization with HEL-OVA (380 and 390 measurements from 3 experiments). b, c, d, Individual MD4 B cells were tracked for their cumulative interactions with OT-2 T cells over 3 hours. b, Examples of tracked MD4 cells (arrowheads). The MD4 cells represented by white tracks divided during the imaging period, giving rise to two daughter cells represented by red tracks. c, Interaction histories of the MD4 cells in (b) are plotted in the line-box graphs. Vertical lines indicate the periods during which the B cells were present in the imaged volume. Arrows indicate cell divisions giving rise to daughter cells. Each box represents one contact with a T cell, and its height indicates the contact duration (see also Supplementary Movie 5). d, A total of 82 MD4 cells in each condition were tracked in 3 experiments. Each MD4 B cell is plotted as one bar. The bar height is the sum of all T cell contacts (top) or only those contacts that were not shorter than 3 minutes (bottom), a cut-off differentiating cognate from non-cognate T-B interactions with a 95% confidence as determined in (a). e, f, MD4 B cells (7-AAD−CD19+IgMa+CD4−GFP− singlet events) and OT-2 T cells (7-AAD−CD19−CD4+GFP+ singlet events) were analyzed by FACS in 3 experiments at 96 hours post HEL-OVA immunization. Individual symbols: mean of 3–4 mice/group/experiment; Lines: mean of the 3 experiments. e, Cell expansion. Top, numbers of MD4 cells recovered after co-transfer of sap+/+ or sap−/− OT-2 T cells were normalized against the number obtained where no exogenous OT-2 cells were co-transferred (dotted line at unit 1). Bottom, absolute numbers of recovered OT-2 cells under the condition of co-transfer with B cells. f, Surface expression of CXCR5, CD40L, ICOS, and OX40 by the two types of OT-2 T cells. See Online Methods for details of quantitative analysis and Supplementary Fig. 4 for representative cytometry plots.