Fig 1.
The recall alloantibody response is B cell intrinsic. (A) Antibody responses following primary and secondary immunization with BALB/c spleen cells (s.c.). Donor specific antibodies (DSA) were measured by flow cytometry (mean fluorescence intensity (MFI)) and anti-Kd antibodies by ELISA. N=5 mice per experiment, repeated twice. (B) Adoptive transfer to demonstrate memory B cell responses. C57BL/6 mice (IgHb) were immunized with BALB/c splenocytes (s.c.), and ≥10 weeks later, mice were sacrificed and B cells were purified and adoptively transferred ((20 × 106) into congenic IgHa recipients. One day after transfer, IgHa recipient mice were immunized with BALB/c splenocytes, and 7 days later, donor specific IgMb and IgG1b produced by transferred B cells, and IgMa and IgG1a produced by recipient B cells, were quantified by flow cytometry. N=2–3/group, repeated 2 times. (C) Gating strategy and percentage of IgG+ B cells and H-2Kd-binding IgG+ B cells from naïve or sensitized mice (left two panels), and phenotype of memory H-2Kd-binding IgG+ versus IgG− B cells. Representative histograms and MFI for CD73, CD38 and CD273 expression are presented (right two panels). N=3–4/group, repeated 3 times. (D) Total number of IgG memory and H-2Kd-binding IgG+ cells per sensitized or naïve mouse (pooled spleen plus 6 lymph nodes). (E) Co-expression of CD273 and CD73 on memory H-2Kd-binding IgG+ B cells.