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Figure S1: Alloanergization with belatacept-mediated CD28 costimulatory blockade induces alloantigen-specific hyporesponsiveness. (A) Thymidine incorporation proliferation assays demonstrate induced responder alloproliferation as a result of coincubation of responder PBMCs with irradiated stimulator PBMCs from unrelated donors in the absence (RS) or presence (alloanergized [RSB]) of belatacept for 5 days with tritiated thymidine added during the last 18 h of culture. Reduction of tritiated thymidine incorporation was measured with belatacept CSB (p = 0.008, median 91% inhibition of the primary alloresponse). Horizontal bars show the median CPM values of the recovered allostimulated versus alloanergized wells. (B) Bar graphs demonstrate the retention of normal mitotic response to anti-CD3 + anti-CD28 stimulation in RSB cells (gray bars) as compared to RS cells (white bars). Bars denote the mean and standard deviation of tritiated thymidine incorporation as CPM. (C) Following the removal of belatacept and alloantigen restimulation (secondary MLR) by a second round of irradiated first party stimulators, alloanergized cells maintain hyporesponsiveness (p = 0.008) as measured by thymidine incorporation during the last 18 h of a total of 8 days in culture. Horizontal bars denote the median CPM values of the recovered allostimulated versus alloanergized wells following restimulation subsequent to CSB removal. (D) Similar to (B) above, RS and RSB anti-CD3/CD28 proliferative responses are retained in the secondary MLR culture.

Figure S2: (A) Allorestimulation of alloanergized human PBMCs also increases the proportion of CD28 CD8+ cells when B7 antibodies are used during alloanergization. Frequencies of CD28 cells, expressed as a proportion of CD8+ cells, are shown as box plots depicting median, 25th and 75th centiles and maximum and minimum values in baseline (untreated) PBMCs and in HLA-mismatched allostimulated and alloanergized cells and after one round of allorestimulation using first party allostimulators. Aggregate data from six experiments with unique stimulator–responder pairs are shown. (B) Allorestimulation of alloanergized human PBMCs also increases the proportion of CD28 CD8+ cells in the haploidentical setting. Frequencies of CD28 cells, expressed as a proportion of CD8+ cells, are shown as box plots depicting median, 25th and 75th centiles and maximum and minimum values in baseline (untreated) PBMCs and in haploidentical allostimulated and alloanergized cells and after one round of allorestimulation using first party allostimulators. Aggregate data from four experiments with unique stimulator–responder pairs are shown.

Figure S3: Repeat rounds of allorestimulation results in relative and absolute expansions of CD8+FOXP3+ cells. (A) Dot plots from a representative experiment showing FOXP3 expression on CD8+ cells in untreated, alloanergized and alloanergized allorestimulated PBMCs. Values shown in boxes represent the percentages of CD8+ cells that are FOXP3 positive. (B) Aggregate data from four experiments. FOXP3+ cells, expressed as a proportion of CD8+ cells, are shown as box plots depicting median, 25th and 75th centiles and maximum and minimum values. (C) Absolute numbers of CD8+FOXP3+ cells in untreated and alloanergized cells following two rounds of first party allorestimulation. Aggregate data from nine experiments. Box plots depict median, 25th and 75th centiles and maximum and minimum values.

Figure S4: CD8+CD28 and CD8+CD28+ cells from allorestimulated alloanergized cultures do not reduce functional anti-CMV responses. (A) Dot plots from a representative experiment showing FOXP3 expression on CD8+ cells in untreated, alloanergized and alloanergized allorestimulated PBMCs. Values shown in boxes represent the percentages of CD8+ cells that are CD107a positive. (B) Aggregate data from four experiments in which autologous responders were stained for CD8 and CD107a after being unstimulated (Mock), or stimulated with CMV lysate or SEB, in the absence of any added cells (A, first row), or with 1:16 ratio of CD8+CD28 (middle row), or CD8+CD28+ (bottom row). CD107a+ cells, expressed as a proportion of CD8+ cells (B), are shown as box plots depicting median, 25th and 75th centiles and maximum and minimum values, with p-values from nonparametric Mann–Whitney tests.

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