Changes in the T-cell repertoire in patients with or without IRAE following treatment with ipilimumab plus GM-CSF. A, Flow cytometry was used to measure the percentage of Ki67+ expression in CD4+ CD3+ FOXP3− lymphocytes (top row) or from CD4− CD3+ lymphocytes (bottom row), either before (week 0, left column) or after treatment (week 2, right column). A representative mCRPC patient who received ipilimumab plus GM-CSF is shown. Statistical analysis was done both on percentages and absolute numbers of Ki67+ lymphocytes. B, The frequency distribution of unique TCR clonotypes is shown for one representative patient who developed an IRAE (IRAE; top plot) and one representative patient who did not develop an IRAE (non_IRAE; bottom plot). The x-axis represents each unique clonotype in descending order of frequency, and the log10 of the frequency for each clonotype at week 0 (black) and week 2 (red) on the y-axis. C–H, The number of unique clones (C, E, and G) and clonality (D, F, and H) of TCR from PBMCs either before (week 0) or 2 weeks after treatment with ipilimumab (week 2) was calculated for all patients (C and D), IRAE patients (E and F), or non_IRAE patients (G and H). The median and interquartiles are shown. *, P < 0.05; **, P < 0.01 by paired Wilcoxon test. IRAE patients exhibit a significant decrease in clonality with treatment. Only data from patients who had samples at both timepoints are shown (n = 21 total patients, 8 IRAE, 13 non_IRAE).
T-cell clonotype expansion and mobilization of newly detected T-cell clones following treatment with ipilimumab plus GM-CSF. Top, fraction of clones, which were found only at week 2 (Week 2 only), only at week 0 (Week 0 only), or at both timepoints (Both) for all patients (A), and separated by IRAE and non_IRAE patients (B). Compared with non_IRAE patients, IRAE patients had a significantly higher fraction of clones that were newly detected at week 2, and a lower fraction of clones that were only found at week 0 (baseline). C and D, T-cell clonotype changes from week 0 to week 2 were binned by fold change in clonal frequency into “increased” (greater than 4-fold change), “decreased” (less than 0.25-fold change), or “unchanged” (between 0.25- and 4-fold change). The analysis is shown for all patients (C) and separated by IRAE and non_IRAE patients (D). The median and interquartiles are shown. IRAE patients also showed a significant increase in the fraction of clones whose frequencies increased with treatment with ipilimumab plus GM-CSF compared with non_IRAE (*, P < 0.05). There was also a borderline significant reduction in the fraction of clones, which decreased with treatment in the IRAE group (P = 0.05, bar not shown). All P values were calculated by two-sample Wilcoxon test.
Timing of changes in clonality and the development of toxicity or clinical response. A, The relative clonality (i.e., the ratio of clonality at each posttreatment timepoint to the clonality at the immediately preceding timepoint) is shown for IRAE and non_IRAE patients for posttreatment weeks 2, 4, 6, 8, and 12. The median and interquartiles are shown. A dotted line at a ratio of 1.0 indicates the relative clonality at which the earlier and later clonality indices were identical, that is, there was no difference in diversity between successive timepoints. Significant decreases in the relative clonality were seen in IRAE patients compared with non_IRAE patients at week 2 (P value for relative clonality of IRAE vs. non_IRAE: 0.045 for week 2/week 0 by two-sample Wilcoxon test). B, Clonality over time is shown for a patient who was treated with four planned doses of ipilimumab (open arrows) plus scheduled GM-CSF, and then later developed an IRAE (panhypopituitarism, onset indicated with filled arrow), which was treated with steroids (gray bar), but also had an exceptional clinical response (PSA decline >90%) to therapy. Clonality (black) is plotted against serum PSA levels (red). The early increase in diversity (i.e., drop in clonality) preceded the subsequent development of AEs and clinical response, and there was no marked change in clonality at the time of IRAE development or PSA decline. C, Clonality at weeks 0 and 2 for PSA responders (>50% decline in PSA, left) versus nonresponders (right) are shown. *, P < 0.05 by two-sample Wilcoxon test. PSA responders specifically show a significant decline in clonality with treatment. The median and interquartiles are shown.
Changes in CD4+ and CD8+ T cells after ipilimumab plus GM-CSF. A, Clonality over time is shown for specific TCR clones, which were identified in sorted CD4+ and CD8+ cells from individual patients and then mapped to bulk PBMCs from all available timepoints in the same patients. Data from available patients who had baseline week 0 data is shown (two IRAE patients, shown in red; one non_IRAE patient, shown in black). B, Flow cytometry was used to quantitate CD4+ FOXP3+ Treg cells. Data including gating from a representative timepoint are shown. Absolute numbers of Tregs are shown for all patients (C) and separated by IRAE (red) and non_IRAE (black; D).
NOTE: Shown are median values with interquartile percentages, as well as P values for the comparison of relative clonality (week 2/week 0) for patients with or without AEs (two-sample Wilcoxon test) when specific AEs are added to the IRAE category as a test of the magnitude of their effect on clonality. The addition of temporal arteritis to the IRAE category increases the significance of difference in clonality between patients with or without AEs.
↵aP value was calculated for the comparison of relative clonality (week 2 divided by week 0) for AE and non-AE groupings listed using two-sample Wilcoxon test. Underline indicates the degree of significance.