CTLA-4 Blockade following Relapse of Malignancy after Allogeneic Stem Cell Transplantation Is Associated with T Cell Activation But Not with Increased Levels of T Regulatory Cells

(A) Lymphocyte phenotypes of patients at baseline and normal controls. The percentages of CD3⁺, CD4⁺, and CD45RO⁺ T cells are significantly lower in patients than in the controls (P = .036, <.0001, and .032, respectively). (B) Absolute numbers of lymphocyte subsets in patients before and after ipilimumab infusion at day 60. CD19⁺ B cells were excluded in 2 cases of CLL to reduce the huge variation that this would cause. The cell counts in all T cell subsets were increased significantly from day 0 to day 60 (Wilcoxon’s signed-rank test; P < .01 for the T subsets). Results are presented as mean ± SD.

Comparison of changes in CD4⁺ and CD8⁺ T cell counts in two groups of patients before and after ipilimumab infusion. (A) On day 0, although most patients in the lower-dose group had lower CD4⁺ T cell counts than the patients in the higher-dose group, the difference was not statistically significant (P = .09). After ipilimumab infusion, the CD4⁺CD3⁺ T cell count was significantly increased in the higher-dose group from day 0 to day 60 (P = .049). (B) On day 0, the absolute cell count of CD8⁺CD3⁺ T cells did not differ in the two groups of patients (P = .52), and there was no significant change in either group after ipilimumab infusion.

(A) Flow cytometry analysis of CD4⁺CD25^high Treg cells from two patients (designated A and B) shown in density plots of the CD4⁺ T cell region. In patient A, two regions in CD4⁺ T cells were identified: CD25^neg and a tail-like CD25^high, without a clear-cut region of CD25^low. This pattern was identified in 9 of 29 patients as well as in 23 of 26 normal controls. In patient B, 3 regions in CD4⁺ T cells were identified based on cell density: CD25^neg, CD25^low, and a tail-like CD25^high. MFI indicated the CD4 expression for each gated cell region, which was lower in CD4⁺CD25^high T cell regions than in CD25^low and CD25^neg cell regions. This pattern was identified in 20 of 29 patients. (B) Expression of CD4⁺CD25^high T subset in CD4⁺ cells and absolute cell counts in 29 patients before and after ipilimumab infusion. There was a significant decrease in the percent expression of the CD4⁺CD25^high T subset from baseline to day 60 (P = .04), but no change in the absolute cell count.

Flow cytometry analysis of intracellular Foxp3 and CTLA-4 expression in CD4⁺ T cells. The density plot analysis gated on CD4⁺ T cells from one patient on day 0 and on day 7 after ipilimumab infusion. The percentage of CD4⁺CD25^highFoxp3⁺ cells was similar to the percentages of CD4⁺CD25^high and CD4⁺CD25^highCTLA-4⁺ T cells and did not change after treatment. Foxp3 also was expressed in CD4⁺CD25^lo/- cells, which were slightly increased in this case after treatment. CTLA-4 expression in CD4⁺CD25^lo/- T cells increased 3-fold from baseline to day 7.

Expression of the CD4⁺CD25^highFoxp3⁺ T subset in CD4⁺ cells and the absolute cell count from the later 11 patients before and after ipilimumab infusion. A significant decrease in the percentage expression in the CD4⁺CD25^highFoxp3⁺ T subset is seen, (P = .02), but no change in absolute cell count.

(A) Comparison of Foxp3 and CTLA-4 expression in CD4⁺ T cells in patients (n =11) and normal controls (n = 12). The percentages of Foxp3 and CTLA-4 in both CD25^lo/- and CD25^high T cells were significantly higher in the patients than in the controls (P < .005). (B) The absolute cell counts of Foxp3⁺ and CTLA-4⁺ in CD4⁺CD25^high T cells did not change at day 60, but CD4⁺CD25^lo/-CTLA-4⁺ T cell counts were significantly increased at day 60 (P = .001).

Analysis of activated T cells in patients receiving higher doses of ipilimumab. The absolute counts of CD4⁺CD69⁺, CD8⁺CD69⁺, and CD4⁺CD25^low T cells in the last 15 patients and of CD4⁺HLA-DR⁺ cells in the last 10 patients are shown before and after ipilimumab infusion. CD4⁺HLA-DR⁺ and CD4⁺CD25^low T cell counts increased significantly from baseline to day 60 (P =.04 and .002, respectively), but CD69⁺ T cell counts did not change significantly.