CD4⁺CD25⁺FoxP3⁺Tregs protect against antibody-mediated acute lung injury. (A-B) Lung W/D weight ratios of untreated (−) BALB/c mice or mice that first underwent in vivo depletions of T cells (A) or B cells (B) by the indicated antibody injections (+) and were then injected with the TRALI-inducing antibody 34-1-2s or indicated control antibodies. (C) Representative flow cytometric dot plot analysis of spleen, thymus, peripheral blood, and lung cells showing the extent of a typical in vivo depletion of CD4⁺CD25⁺FoxP3⁺ Tregs (splenic cells were depleted from 9.82% to 2.4%, thymic cells were depleted from 0.59% to 0.1%, peripheral blood cells were depleted from 2.52% to 0.2%, and lung cells were depleted from 3.7% to 0.5% of total CD4⁺ T cells). (D) Representative macroscopic images of lungs from a Treg-depleted mouse 90 minutes after injection of the indicated control antibody (left) or 34-1-2s (right). (E) Lung W/D weight ratios of CD4⁺CD25⁺FoxP3⁺ Treg-depleted BALB/c mice injected with the indicated isotype control antibodies and/or 34-1-2s. (F) Lung histology from BALB/c mice receiving the indicated antibody injections: subpanels A1-E1 and A2-E2 represent lung tissue images taken at magnification ×20 and ×40, respectively. Representative images of each indicated group are shown. A zoom of indicated square in E2 is depicted alongside and shows alveolar PMN infiltration. Scale bars represent 100 µM in subpanels A1-E1 and 50 µM in subpanels A2-E2. (G) Measurement of tissue elastance in Treg-depleted BALB/c mice that received either 34-1-2s injection or indicated isotype control. All mice were analyzed 90 minutes after the second injections. For the statistical analyses, only significant comparisons of interest are shown. Panel A was analyzed with a 1-way ANOVA with a Tukey’s post hoc test, panel E was analyzed with a 1-way ANOVA with Dunn’s post hoc test, and panel G was analyzed by a 1-tailed Mann-Whitney test. Each dot represents 1 mouse, and error bars represent SD. *P < .05, ***P < .001, ****P < .0001. IgG, immunoglobulin G; NS, nonsignificant.