Fig. 1

Time-dependent response of exogenous mature naïve B cells at the site of CCI injury. A Schematic representation of injury and treatment paradigm. B cells are injected intraparenchymally at the time of CCI, and both injured (ipsilateral) and contralateral brain hemispheres are collected for analysis at determined intervals. B At 18 h after CCI, exogenous CD45.1⁺ CD22⁺ B cells injected at the time of lesion remain aggregated at the site of tissue injury, while infiltrating CD11b⁺ myeloid cells surround and intersperse with the B cells (arrows). C Exogenous CD45.1⁺ B cells can be readily identified in flow cytometry analysis of dissociated tissue from treated mice, in both injured and sham-injured brain hemispheres. By contrast, saline-treated controls show minimal B cell infiltration. Sample results are shown at 18 h post-injury and treatment. D The number of B cells at the injury site decreases steadily over time, and by 2 months post-CCI and treatment, no B cells could be observed in the biopsies collected from the injury site. E–L Increasing proportions of the retrieved populations of B cells injected into the injured brain microenvironment became activated over time, producing both pro- and anti-inflammatory cytokines. Note that while inflammatory cytokines such as IL-6, IFNγ, or TNFα peak early after CCI injury, a large proportion of the exogenous B cells continue to produce regulatory cytokines, including IL-35, IL-10, and TGFβ, persistently, at least up to 10 days after administration. Statistical significance was assessed by one-way ANOVA, followed by Dunnett's multiple comparisons test against the sham-injury control. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. M Minimal activation and cytokine production was observed in the control conditions, when B cells were maintained on ice or exposed to an uninjured brain microenvironment. N By contrast, the majority of B cells injected at the lesion site begin producing a combination of cytokines by 18 h, peaking at 48–96 h post-injury. tSNE analysis indicated that there was a substantial overlap between the source cells for a variety of pro- and anti-inflammatory cytokines. By 4 days and even more so at 10 days post-application, subsets of B cells that produce exclusively regulatory cytokines, IL-10 and IL-35, become apparent and dominate the B cell population within the injury. In histograms, tSNE counts are normalized to mode