![Figure 2. Animals with breakthrough acute GVHD develop clinical and pathological evidence of disease without the specific T-cell signatures that develop during hyperacute GVHD. (A) Expression of Ki67 and granzyme B (GrmB) in T cells from peripheral blood, measured longitudinally posttransplant. Colored circles represent the clinical categories of hyperacute (red), suppressed (green), and breakthrough acute (orange) disease. Statistical differences between the hyperacute and suppressed cohorts were evaluated at days 2, 5, 8, 12, 18, and 25 posttransplant using an unpaired Student t test, and significant differences of the mean percentage of either CD4 (*) or CD8 (†) cell subtypes were determined using an unpaired Student t test. All other time-point comparisons were not significant. (B) Engraftment/expansion of CD4 and CD8 TCM and TEM from peripheral blood, measured longitudinally posttransplant. Colored circles represent the clinical categories of hyperacute (red), suppressed (green), and breakthrough acute (orange) disease. Statistical differences between the hyperacute and suppressed cohorts were evaluated at days −7, 2, 5, 8, 12, 18, and 25 posttransplant using an unpaired Student t test. *Significant differences of the absolute numbers of the CD4 and CD8 T-cell subpopulations. All other time-point comparisons were not significant. (C) The combined GVHD pathology score (summarizing liver, colon, skin, and lung scores [top panel]) and combined upper GI GVHD score (summarizing esophagus, stomach, and duodenum [Duod.]; [bottom panel]) of recipients from hyperacute (MST = 12 days) and breakthrough acute (MST = 34.5 days) cohorts. Histopathologic analysis was performed at the time of terminal analysis and analyzed by a pathologist (A.P.-M.) in a blinded manner. *P < .05 using the Mann-Whitney test on composite scores. NS, not significant. (D) Flow cytometric analysis of mononuclear cells isolated from peripheral blood, lymphoid, and nonlymphoid GVHD-target organs at the time of terminal analysis from animals from hyperacute and breakthrough acute cohorts and stained for Ki-67 (top and middle panel) and for granzyme B (bottom panel). Plots depict the percentages of CD4 and CD8 T cells expressing Ki-67 and the percentage of CD8 T cells expressing high levels of granzyme B (mean fluorescence intensity [MFI] ≥ 105). *P < .05 using the Mann-Whitney test. LN, lymph node.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/128/21/10.1182_blood-2016-07-726547/4/blood726547f2.jpeg?Expires=1723436289&Signature=PZcuiJlWNtWYsnL5~0L3MfZDTvff00ESItQNU4seYhoGy45aI9v67~lUfblyLvTt6QAqol8VsbfD-MVFU7MrZcuEsLVt20Ts-kaW-jyT4xOJ1w513AN4qRYOHLT3OPLWeKKzbxJpNlWveqghPejuU01mmAEBMe~nI2CZBknkZe4xs5kEP3wQJtHASd76kP2fRQEbAxlCDGKQ-z1b1Iao0en5eaNpmgxBvzZK2KB-LghAA2vPXiehLGvBc2p1T5yvNDcDhe4zmHupfHWPlyvOK61n1NpiD5Ve8i19a0ZmxCm0gVzuIF7hGpiSsXplo5q7q6Hd3z8zZh80zv9eQnoqig__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Animals with breakthrough acute GVHD develop clinical and pathological evidence of disease without the specific T-cell signatures that develop during hyperacute GVHD. (A) Expression of Ki67 and granzyme B (GrmB) in T cells from peripheral blood, measured longitudinally posttransplant. Colored circles represent the clinical categories of hyperacute (red), suppressed (green), and breakthrough acute (orange) disease. Statistical differences between the hyperacute and suppressed cohorts were evaluated at days 2, 5, 8, 12, 18, and 25 posttransplant using an unpaired Student t test, and significant differences of the mean percentage of either CD4 (*) or CD8 (†) cell subtypes were determined using an unpaired Student t test. All other time-point comparisons were not significant. (B) Engraftment/expansion of CD4 and CD8 TCM and TEM from peripheral blood, measured longitudinally posttransplant. Colored circles represent the clinical categories of hyperacute (red), suppressed (green), and breakthrough acute (orange) disease. Statistical differences between the hyperacute and suppressed cohorts were evaluated at days −7, 2, 5, 8, 12, 18, and 25 posttransplant using an unpaired Student t test. *Significant differences of the absolute numbers of the CD4 and CD8 T-cell subpopulations. All other time-point comparisons were not significant. (C) The combined GVHD pathology score (summarizing liver, colon, skin, and lung scores [top panel]) and combined upper GI GVHD score (summarizing esophagus, stomach, and duodenum [Duod.]; [bottom panel]) of recipients from hyperacute (MST = 12 days) and breakthrough acute (MST = 34.5 days) cohorts. Histopathologic analysis was performed at the time of terminal analysis and analyzed by a pathologist (A.P.-M.) in a blinded manner. *P < .05 using the Mann-Whitney test on composite scores. NS, not significant. (D) Flow cytometric analysis of mononuclear cells isolated from peripheral blood, lymphoid, and nonlymphoid GVHD-target organs at the time of terminal analysis from animals from hyperacute and breakthrough acute cohorts and stained for Ki-67 (top and middle panel) and for granzyme B (bottom panel). Plots depict the percentages of CD4 and CD8 T cells expressing Ki-67 and the percentage of CD8 T cells expressing high levels of granzyme B (mean fluorescence intensity [MFI] ≥ 105). *P < .05 using the Mann-Whitney test. LN, lymph node.
