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

Article Outline

• Abstract
• Introduction
• Patients and Methods
  • Patients
  • Lymphocyte Count
  • Antibodies
  • Flow Cytometry
  • Statistical Analysis
• Results
  • Flow Cytometry Analysis
    • Lymphocyte subset analysis
    • CD4+CD25high Treg cells
    • Detection of Foxp3 in CD4+ T cells
    • Detection of CTLA-4 in CD4+ T cells
    • Expression of T Cell activation markers
  • Correlation with Clinical Findings
• Discussion
• Acknowledgments
• References
• Copyright

Cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) is a key negative regulator of T cell activation and proliferation. Ipilimumab is a human monoclonal antibody that specifically blocks the binding of CTLA-4 to its ligand. To test the hypothesis that blockade of CTLA-4 by ipilimumab could augment graft-versus-malignancy (GVM) effects without a significant impact on graft-versus-host disease (GVHD), we conducted a phase I clinical trial of ipilimumab infusion in patients with relapsed malignancy following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Here, we report the analysis of peripheral blood T lymphocyte reconstitution, T regulatory cell (Treg) expression, and T cell activation markers after a single dose of ipilimumab in 29 patients. Peripheral blood samples were collected from all patients before and after ipilimumab infusion. Lymphocyte immunophenotyes, including levels of CD4⁺CD25^high cells and T cell activation markers, were analyzed in all cases. Levels of CD4⁺CD25^highFoxp3⁺ cells and intracellular CTLA-4 in CD4⁺ T cells also were evaluated in the last 11 cases. We found lower baseline levels of CD4⁺ and CD45RO⁺ T cells in patients compared with normal controls. More than 50% of the patients had abnormally low lymphocyte counts (CD4 or/and CD8 T cells), and some had no circulating B lymphocytes. The percentages of both CD4⁺CD25^high and CD4⁺CD25^highFoxp3⁺ T cells were significantly higher in patients before ipilimumab infusion than in healthy donors. Twenty of 29 patients exhibited an elevated level of CD4⁺CD25^low activated T cells at baseline, compared with only 3 of 26 healthy donors. Both CD4⁺ and CD8⁺ T lymphocyte counts were significantly increased after ipilimumab infusion. There was no consistent change in absolute lymphocyte count or in the number of T cells expressing the activation marker CD69. However, increases in CD4⁺CD25^low T cells were seen in 20 of 29 patients and increases in CD4⁺HLA-DR⁺ T cells were seen in the last 10 patients in the first 60 days after ipilimumab infusion. Although the percentages of both CD4⁺CD25^high and CD4⁺CD25^highFoxp3⁺ T cells decreased significantly during the observation period, the absolute cell counts did not change. Intracellular CTLA-4 expression in CD4⁺CD25^lo/- T cells increased significantly after ipilimumab infusion. We conclude that CTLA-4 blockade by a single infusion of ipilimumab increased CD4⁺ and CD4⁺HLA-DR⁺ T lymphocyte counts and intracellular CTLA-4 expression at the highest dose level. There was no significant change in Treg cell numbers after ipilimumab infusion. These data demonstrate that significant changes in T cell populations occur on exposure to a single dose of ipilimumab. Further studies with multiple doses are needed to explore this phenomenon further and to correlate changes in lymphocyte subpopulations with clinical events.

Key Words: Ipilimumab, T lymphocytes

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Introduction 

Relapse of malignancy after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a major obstacle to treatment success [1]. Conventional treatment of relapse following allo-HSCT is usually unsuccessful, and most patients eventually succumb to their malignancy. The exact mechanism behind the failure of adoptive immunotherapy after allo-HSCT is unclear but might include a lack of specific immune activation, lack of cancer-specific antigens, poor antigen presentation to donor immune cells, and a relative paucity of alloreactive lymphocytes compared with the numbers of proliferating cancer cells 1, 2.

Regulatory T (Treg) cells are generated in the thymus and function as immunosuppressive regulators. They are best defined as a subset of CD4⁺ T cells with a phenotype of CD25⁺ and Foxp3⁺, and usually account for <5% of the total CD4⁺ T cells in the peripheral blood 3, 4. Cytotoxic T lymphocyte antigen 4 (CTLA-4) is expressed on effector T cells following antigen-specific activation, where it functions as a key negative regulatory factor. It is also constitutively expressed on the Treg cell surface [5]. Identification of Treg cells has remained controversial because of the lack of Treg-specific markers that separate this lymphocyte subpopulation from activated T effector cells [6]. Treg cells play a critical role in maintaining immune tolerance and regulating graft-versus-host disease (GVHD) and graft-versus-malignancy (GVM) effects. The mechanisms of immune suppression regulated by Treg cells have been found to require cell contact between Treg and effector cells as well as cytokines, such as interleukin (IL)-10 and transforming growth factor β [7]. A recent report in a mouse model found that Treg cells mediated suppression of GVHD and GVM through different mechanisms [8]. GVHD suppression did not require granzyme B, whereas previous studies had shown that granzyme B was involved in suppression of antitumor responses. Malignant cells can recruit Treg cells locally to suppress T cell function and create a favorable microenvironment for tumor cell growth 9, 10, 11. Clinical studies have shown an increased number of Treg cells in tumor sites, peripheral blood, and tumor-infiltrated lymph nodes from both solid tumors and hematologic malignancies [12]. This increased expression of Treg cells has been associated with poor clinical outcomes [13]. The development of two CTLA-4 monoclonal antibodies, ipilimumab and tremelimumab, has made CTLA-4 an attractive target for cancer immunotherapy. Clinical trials using ipilimumab or tremlimumab as monotherapy or in combination with vaccines, cytokines, or chemotheraputic reagents have been performed in patients with metastatic melanoma, renal cell carcinoma, non-Hodgkin lymphoma, and prostate, colon, and ovarian cancer 14, 15, 16, 17. Objective response rates have been clearly related to antibody dose. The antitumor response is often associated with immune-related adverse events (IRAEs). CTLA-4 blockade–mediated immune responses are associated with tumor-specific cytotoxic T cell activation and expansion. Clinical trials using CTLA-4 blockade found an increase of Th1 cytokines in patients’ plasma and an increase in HLA-DR⁺ CD4⁺ or CD8⁺ T cells, whereas the changes in CD4⁺CD25⁺ or CD4⁺Foxp3⁺ T subsets, defined as Treg cells, were not consistent 18, 19, 20. The increase in activated T cells in the circulation was highly correlated with antibody dose in combination with granulocyte macrophage colony-stimulating factor in a trial of patients with metastatic prostate cancer [21]. In preclinical studies, CTLA-4 blockade was found to modulate Treg cell function without reducing Treg cell numbers, and also to induce both CD4 and CD8 T cell activation in murine models 22, 23, 24.

Treg cells may have several roles after allo-HSCT. The importance of Treg cells in the prevention of acute GVHD (aGVHD) after allo-HCT has been reported 25, 26. The correlation of Treg cell numbers and Foxp3 mRNA expression with chronic GVHD (cGVHD) after allo-HSCT is not clear, however 27, 28. The roles of Treg cells in immune reconstitution, maintenance of the balance between GVHD and GVM, and such treatments as donor lymphocyte infusion and immunosuppressive drugs are not well understood. Clinical studies have shown that selective depletion of T lymphocytes, such as CD25⁺ T cells, in vitro can effectively reduce the risk of aGVHD and cGVHD, but there are disadvantages, including poor engraftment, increased risk of relapse, and delayed immune reconstitution leading to serious infectious complications. Attempts have been made to separate the GVHD and GVM effects; however, clinical trials in humans have shown that the GVM effect and long-term survival are highly associated with the presence of GVHD [29].

We have completed a clinical trial of CTLA-4 blockade in patients with relapse of malignancy after allo-HSCT. This trial was based on the hypothesis that augmenting the immune response to cancer cells by blocking negative regulatory signals might improve the GVM effect. This trial was facilitated by the advent of a fully human monoclonal antibody capable of blocking the interaction of CTLA-4 and Treg cells and the interaction of ligands CD80/86 and antigen-presenting cells. An escalating dose of ipilimumab was given as a single i.v. infusion. The clinical results of this study were reported previously [30]. Here, we report the immunophenotypes of peripheral blood T cells, including T cell reconstitution, activation, and Treg expression, in 29 patients before and after a single-dose infusion of ipilimumab.

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Patients and Methods 

Patients 

A total of 29 patients with relapsed malignancy after allo-HSCT were enrolled in this trial, including 14 with Hodgkin disease, 6 with multiple myeloma, 2 with acute myelogenous leukemia, 2 with chronic lymphocytic leukemia (CLL), 2 with chronic myelogenous leukemia, 1 with renal cell carcinoma, 1 with breast cancer, and 1 with non-Hodgkin lymphoma. The first 17 patients were enrolled in the dose-escalation phase of the study, and the last 12 patients were in the phase II portion of the study. The median time from transplantation to enrollment in the study was 21 months (range, 4-79 months). Characteristics of the patients were published previously [30]. Before the trial, 3 patients had extensive cGVHD, 4 patients had limited cGVHD, and 3 patients had grade I/II aGVHD. Only 2 patients remained in limited cGVHD at trial entry. In all patients, immunosuppressive drugs had been discontinued for more than 6 weeks. Twenty-six patients had progressive disease, 2 patients had stable disease, and 1 patient was in remission after 2 months of imatinib therapy. Most patients had 100% donor chimerism in both T cells and myeloid cells. Ipilimumab (Medarex, Bloomsbury, NJ) was given as a single i.v. infusion with a dose escalating from 0.1 mg/kg to 3 mg/kg. No other immunomodulatory therapy was given during the 60-day observation period. Peripheral blood samples from all patients were obtained before ipilimumab infusion (day 0) and on days 7, 14, 30, and 60 after ipilimumab infusion. Blood samples were obtained on days 1 and 3 from the later cohort of 14 patients.

Peripheral blood from 26 healthy individuals was obtained from the San Diego Blood Bank and used as normal controls. The peripheral blood lymphocytes from 12 donors were separated and cultured with IL-2 (200 μ/mL) for 3 days and used as controls for T cell activation markers.

Lymphocyte Count 

The absolute lymphocyte count (ALC) was calculated based on the percentage of lymphocytes in automated complete blood counts from peripheral blood. Absolute cell count was calculated as ALC × percentage of expression of markers for T subset or B lymphocytes by flow cytometry analysis.

Antibodies 

A panel of fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, or peridinin chlorophyll protein complex (PerCP)-labeled antibodies, including CD3, CD4, CD8, CD11a, CD16, CD19, CD25, CD38, CD45RA, CD45RO, CD56, CD62L, C69, CD152 (CTLA-4), TCRαβ, TCRγδ, and HLA-DR, and the isotype controls, were purchased from BD Biosciences (San Jose, CA). T regulatory cell staining kits, including CD4 FITC, CD25 PE, and Foxp3 PE-cy5 (clone PCH102), and staining buffer sets were purchased from eBioscience (San Diego, CA).

Flow Cytometry 

Peripheral blood mononuclear cells (PBMCs) were obtained using a standard Ficoll-Hypaque density-gradient centrifugation. Cells were stained with a panel of antibodies and analyzed using FACScan and Cellquest software (BD Biosciences, San Jose, CA). For analysis of intracellular Foxp3 expression, cells were fixed after surface staining with CD4 FITC and CD25 PE, washed with a permeabilization buffer, and then stained with Foxp3 PE-cy5 or isotype control. For analysis of CD4⁺CD25⁺ and CD4⁺CD25⁺Foxp3⁺ T cells, the CD4⁺ cell region was first gated using forward scatter versus FL1 (FITC) or FL3 (PerCP) in a dot plot. The CD4⁺CD25^high and CD4⁺CD25^low regions were then analyzed in a density plot. Treg cells were identified as a CD25 and Foxp3 dual-positive population. The expressions of T cell activation markers, including HLA-DR, CD69, and CD25, were analyzed in either CD4⁺ or CD8⁺ T cell regions.

Statistical Analysis 

For baseline values or changes from baseline, the Mann-Whitney U test was used for between-group comparisons, and Wilcoxon’s signed-rank test or the paired t test was used for within-group comparisons. For data collected over several time points, mixed-effects models were used to test for a significant within-group change over time, adjusted for such covariates as age at diagnosis and type of diagnosis. A random intercept was used to account for correlation of the data for each patient, and conditional studentized residuals were plotted to check model fit. Pearson’s correlation tests were used to test for a nonzero correlation between two variables. Analyses were performed using Prizm 5 (Graphpad Software, San Diego, CA) and SAS version 9.2 (SAS Institute, Cary, NC). All tests were two-sided at the 5% significance level.

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Results 

Flow Cytometry Analysis 

Eighteen patients had lymphocytopenia, defined as an ALC <1500/μL in peripheral blood at baseline. Six of these patients had both CD4⁺ and CD8⁺ T cell counts <200/μL, and 10 patients had either a CD4⁺ or CD8⁺ count <200/μL. Five patients had no detectable CD19⁺ B cells. Two patients with relapsed CLL had an ALC >10,000/μL, and 70%-90% of these cells were CD19⁺ monoclonal B cells. Two patients with relapsed acute myelogenous leukemia had a small percentage of circulating blasts. Figure 1A shows the phenotypes of lymphocyte subsets from patients at baseline and healthy individuals. Although the overall pattern of lymphocyte subsets appeared to be similar in the patients and normal controls, the percentages of CD3⁺, CD4⁺, and CD45RO⁺ T cells were significantly lower in the patients. The ratio of CD4 to CD8 T cells was close to 1:1 in the patients, significantly lower that of 2:1 in the controls (P = .01). We analyzed the ALC counts of lymphocyte subsets at day 0 and day 60 in the patients (Figure 1B). All T cell subsets demonstrated significantly increased numbers at day 60. Between day 0 and day 60, CD4⁺ T cell count increased in 19 patients (66%) and CD8⁺ T cell count increased in 23 patients (79%). In the 16 patients with a baseline CD4 or/and CD8 cell count <200/μL, 7 of 10 (70%) had an increased CD4⁺ T cell count and 9 of 11 (82%) had an increased CD8⁺ T cell count. There was no consistent change in the numbers of CD19⁺ B cells or CD56⁺ CD16⁺CD3^neg NK cells from day 0 to day 60 after ipilimumab infusion.

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• Figure 1 

  (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.

We compared the changes in ALC and counts of CD4⁺ and CD8⁺ T cells in two groups of patients, those who received <3 mg/kg of ipilimumab (n = 14) and those who received 3 mg/kg of ipilimumab (n = 15). Figure 2 shows CD4⁺ and CD8⁺ T cell counts from days 0, 7, 14, 30 and 60 in both groups. We found a significant increase in CD4⁺ T cell count from day 0 to day 60 in the patients who received the higher dose. The median change was 95 cells/μL (interquartile ranghe [IQR], 0-170) in the lower-dose group and 260 cells/μL (IQR, 0-360) in the higher-dose group (P = .049). There was no consistent change in ALC and CD8⁺ T cell count between the two groups.

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• Figure 2 

  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.

We initially analyzed CD4⁺CD25^high T cells as Treg cells in all 29 patients and 26 healthy donors by the classical method [31]. Within the CD4⁺ T cell region were 3 cell populations based on CD25 expression by density plot analysis: the CD25^neg region, the CD25^low region, and a tail-like region with low mean fluorescence intensity (MFI) for CD4 expression, defined as the CD25^high region (Figure 3A). At baseline, the percentage of CD4⁺CD25^high T cells was significantly higher in patients compared with healthy donors (median, 7.2% [range, 1.5%-22%] vs 3.2% [0.7%-6.5%]; P < .0001). Eleven of the 29 patients had a CD4⁺ T cell count <200/μL; these patients had a significantly higher baseline percentage of CD4⁺CD25^high T cells compared with the other patients (median, 9.1% [range, 1.2%-22%] vs 6.7% [1.8%-14%]; P = .04). The absolute cell count of CD4⁺CD25^high T cells was 15/μL (range, 1-39/μL) in patients with fewer CD4⁺ T cells and 28/μL (range, 5-116/μL) in those with more CD4⁺ T cells. Figure 3B shows the expression of CD4⁺CD25^high T cells in total CD4⁺ cells and the absolute cell counts from days 0, 7, 14, 30, and 60. In a mixed-effects model adjusted for dose group, age, and diagnosis, in all 29 patients, the percentage of CD4⁺CD25^high T cells decreased significantly over time after ipilimumab infusion (change per month, −0.66%; 95% confidence interval (CI], −1.26% to −0.03%; P = .04), but the absolute CD4⁺CD25^high cell count did not change significantly (P = .10). The model for comparing counts used percent change from baseline as the outcome.

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• Figure 3 

  (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.

When the antibody for Foxp3 became available for flow cytometry studies, we analyzed intracellular levels of Foxp3 and CTLA-4 in CD4⁺ T cells for the last 11 patients (who received a higher dose of ipilimumab) and from 12 healthy donors. Figure 4 shows a representative flow cytometry analysis of a patient before and at day 7 after ipilimumab infusion. At baseline, the percentage of CD4⁺CD25^highFoxp3⁺Treg was significantly higher in patients than in normal controls, accounting for a median of 3.0% (range, 0.4%-7.1%) of CD4⁺ T cells in patients, compared with 0.6% (range, 0.2%-3.9%) in controls (P = .004). The absolute cell counts of CD4⁺CD25^highFoxp3⁺ T cells (median, 11/μL; range 2-65/μL) and CD4⁺CD25^high T cells (median, 15/μL; range, 6-67/μL) were similar at baseline. Foxp3⁺ cells were also found in CD4⁺CD25^lo/- regions (Figure 4). CD4⁺Foxp3⁺ T cells accounted for a median of 4.4% of CD4⁺ T cells (range, 1.7%-10.7%) in patients, compared with 1.4% (range, 0.2%-3.9%) in controls (P = .002). The median absolute cell count of CD4⁺Foxp3⁺ T cells was 10/μL (range, 5-96/μL) at baseline. Figure 5 shows the expression of CD4⁺CD25^highFoxp3 T cells in total CD4⁺ cells and the absolute cell counts from days 0, 7, 14, 30, and 60.

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• Figure 4 

  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.

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• Figure 5 

  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.

From day 0 to day 60 after ipilimumab infusion, the patients demonstrated a significant decrease in the percentages of CD4⁺CD25^highFoxp3⁺ Treg cells (P = .02) and CD4⁺Foxp3⁺ T cells (P = .02), but no significant change in both absolute cell counts (P ≥.90). The model for comparing counts used percent change from baseline as the outcome.

In the last 11 patients analyzed, intracellular CTLA-4 expression was detected in both CD25^high and CD25^lo/- cells, accounting for a median of 3.4% (range, 0.5%-8.6%) of CD4⁺CD25^high T cells and 5.8% (range, 2.3%-12%) of CD4⁺CD25^lo/- T cells at baseline. As shown in Figure 6, the expression of CD25, Foxp3, and CTLA-4 in CD4⁺ T cells was significantly higher in these patients than in the normal controls. The absolute cell count of CD4⁺CD25^highCTLA-4⁺ T cells was comparable to the CD4⁺CD25^highFoxp3⁺ T cell count at baseline (median, 17/μL; range, 1-65/μL). The CD4⁺CD25^lo/-CTLA-4⁺ T cell count was comparable to the CD4⁺Foxp3⁺ T cell count (median, 27/μL; range, 5-99/μL).

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• Figure 6 

  (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).

The intracellular CTLA-4 expression in CD4⁺CD25^lo/- T cells was significantly increased in the 11 patients after ipilimumab infusion (change per month, 6.3%; 95% CI, 2.7%- 9.9%; P = .001), as shown in Figure 6. This increase was seen in almost all 11 patients at day 7 and was sustained to day 60 (data not shown).

We analyzed CD69 expression on both CD4⁺ and CD8⁺ T cells before and after ipilimumab infusion in 29 patients. At baseline, CD69⁺ T cells accounted for a median of 0.8% (range, 0-24%) of total CD4⁺ T cells in patients, compared with 0.3% (range, 0-5%) in normal controls (n = 12; P = .07). For CD8⁺ T cells, the median was 2.1% (range, 0.3%-28%) of CD69⁺ T cells in patients and 2.0% (range, 0.5%-4.3%) in controls (P = .60). After ipilimumab infusion, overall no significant change in CD69 expression was seen in the 29 patients. However, the expression of CD69 on either CD4⁺ or CD8⁺ T cells increased between 3- and 60-fold in 8 patients from day 7 to day 60.

We analyzed the expression of activation marker CD25 on both CD4⁺ and CD8⁺ T cells. CD4⁺CD25^low-activated T cells were detectable in 20 patients (n = 29) at baseline, but in only 3 of 26 controls. Among these 20 patients, these cells accounted for a median of 43% (range, 21%-64%) of the CD4⁺ T cells at baseline. The percentage of CD8⁺CD25⁺ T cells was elevated to 10%-22% of CD8⁺ T cells in 6 patients but in only 1 of 12 controls at baseline. After ipilimumab infusion, no significant change in CD25 expression on CD8⁺ T cells was noted. Although the expression of CD4⁺CD25^low T cells remained stable in all patients at day 60, the cell count significantly increased from day 0 to day 60, from a median of 134/μL (range 23-482/μL) at baseline to a median of 237/μL (range, 46-1197/μL) at day 60 (P = .01), for a daily increase of 2.52 (95% CI, 1.26-3.78; P = .002).

We analyzed CD4⁺ HLA-DR⁺–activated T cells in the last 10 patients enrolled. At baseline, these accounted for a median of 11% (range, 3%-31%) of CD4⁺ T cells in the patients, compared with 4.9% (range, 1%-28%) in normal controls (n = 16) (P = .02). After the ipilimumab infusion, both the percentage expression and the absolute cell count of CD4⁺HLA-DR⁺ T cells were increased significantly in these 10 patients. The absolute cell count increased from a median of 46/μL (range, 6-134/μL) at baseline to 87/μL (range, 41-683/μL) at day 60 (P = .004).

Figure 7 shows the absolute cell counts of CD4⁺CD69⁺, CD8⁺CD69⁺, CD4⁺CD25^low, and CD4⁺HLA-DR⁺ T cells in patients who received an ipilimumab dose of 3 mg/kg before and after antibody infusion.

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• Figure 7 

  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.

Correlation with Clinical Findings 

Clinical response was evaluated monthly by physical examination, computed tomography scan, cytogenetic analysis, polymerase chain reaction, and fluorescein in situ hybridization analysis of bone marrow samples. Three patients exhibited an objective response after ipilimumab infusion [30]. Comparing the responding patients with the nonresponders reveals no significant difference in the ALC and CD4⁺CD25^high T cell counts. At 1 week after ipilimumab infusion, both CD4⁺ and CD8⁺ T cell counts were increased in all 3 responding patients, and activated T cell counts increased in 2 of them.

Clinical GVHD was evaluated monthly in all patients after ipilimumab infusion. No significant change in GVHD status was seen in 2 patients with limited cGVHD and in 8 patients with a previous history of GVHD. Three patients with a history of aGVHD had an ALC <1000/μL at baseline. Eight patients had a high percentage of CD4⁺CD25^high T cells, ranging from 5.4% to 15.7% of total CD4⁺ T cells. Nine patients received a donor lymphocyte infusion after the ipilimumab infusion, and only one patient developed grade I aGVHD.

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Discussion 

Allo-HSCT can cure a number of malignancies through the GVM effect. This effect is even more important when reduced-intensity conditioning is used. However, donor-derived alloreactive T cells can also cause life-threatening GVHD. Activation of T cells requires the recognition of specific antigens as well as costimulatory molecules, such as CD80 and CD86, expressed on antigen-presenting cells. On activation, CTLA-4 is up-regulated and expressed on the T cell surface to provide negative feedback to activated T cells. CTLA-4 is constitutionally expressed on Treg cells. CTLA-4 blockade has been shown to induce antitumor effects in humans with melanoma and certain other malignancies 14, 15, 16, 17, 32, 33, 34.

We studied Treg cells and T cell activation markers in a unique group of patients who underwent ipilimumab therapy for relapsed malignancy after allo-HSCT. The patients received a single dose of ipilimumab infusion in an attempt to augment GVM and induce regression of malignancy. The median time from allo-HSCT to ipilimumab infusion was 21 months. Immune reconstitution was incomplete in more than 50% of the patients, based on low CD4⁺ and CD8⁺ T cell counts, inverted CD4:CD8 cell ratios, and undetectable circulating CD19⁺ B cells. Various factors have a significant impact on immune reconstitution after allo-HSCT, including previous chemotherapy, underlying malignancy, conditioning regimen, GVHD status, immunosuppressive therapy, and the number of Treg cells and their functions. Our clinical trial is the first attempt to study the effect of CTLA-4 blockade on the expression of Treg cells and T cell activation markers in this unique patient population.

We report several interesting observations from this trial. First, almost all patients had a significantly higher percentage of Treg cells (both CD4⁺CD25^high and CD4⁺CD25^highFoxp3⁺ Tregs) compared with the normal controls despite their lymphocytopenia and low CD4⁺ T cell counts. We found that the patients with a CD4⁺ T cell count <200/μL had a higher proportion of CD4⁺CD25^high Treg cells compared with those with a CD4⁺ T cell count >200/μL. Treg cells have been shown to suppress the proliferation, differentiation, and cytokine production of T effector cells. The high percentage of Treg cells might provide a potential explanation for the immune deficiency and relapse of malignancy. The blockade of CTLA-4 on Treg cells might contribute to the increased number of T cells seen in most patients after a single dose of ipilimumab. Clinical studies of patients with myeloma who have undergone allo-HSCT have shown that Treg cell reconstitution occurs earlier and faster than conventional CD4⁺ T cell reconstitution [35]. These CD4⁺CD25⁺Foxp3⁺ Treg cells are donor-derived memory-type T cells that expand primarily in bone marrow. Treg cell reconstitution can occur as early as day 30 posttransplantation in patients who undergo CD25-depleted allo-HSCT. These Treg cells are believed to derive from CD4⁺CD25^- naïve-type T cells by their intracellular Foxp3 expression [36]. Whether there is a correlation between Treg cell frequency and the status of cGVHD, as well as long-term immune reconstitution, is a matter of debate. Without functional analysis, evaluating the potency of the suppressive function of these Treg cells on GVHD and GVM is difficult. A recent report from a multicenter clinical trial of ipilimumab monotherapy in patients with pretreated advanced melanoma (n = 284) showed a dose-dependent efficacy. At a dose of 10 mg/kg, the patients had a significantly higher response rate, although IRAEs were found in all dose ranges, but a large increase in ALC was associated with the highest antibody dose of 10 mg/kg [37]. Increases in both CD4⁺ T cells and CD4⁺Foxp3⁺ Treg cells were reported in a clinical trial using ipilimumab in patients with progressive metastatic hormone-refractory prostate cancer [19]. The Treg cell and T effector cell increases were found to be antibody dose–dependent. It should be noted that Foxp3 can be induced in activated effector T cells [38].

Second, the finding of a high percentage of CD4⁺CD25^low-activated T cells in 20 of 29 patients certainly raises speculation that these donor-derived T effector cells could be functionally activated to mount a GVM attack against the original malignancy. We have seen some evidence of a dose-dependent T cell expansion and increased expression of T cell activation markers, such as HLA-DR, in 10 patients after ipilimumab infusion. The patients who received the 3-mg/kg dose of ipilimumab had significantly higher CD4⁺ T cell counts than those who received lower doses, and significant increases in CD4⁺HLA-DR⁺ activated T cell counts over time were seen in the last 10 patients studied. This finding is similar to results from previous clinical studies of CTLA-4 blockade 39, 40. No significant changes in CD4⁺CD25^high and CD4⁺CD25^highFoxp3⁺ Treg cell counts were seen. Despite the evidence of T cell expansion and activation, we noted no clinically significant immune reaction after ipilimumab infusion, even in the 3 patients who had an objective response.

These data lead us to believe it might be possible to selectively activate the GVM effect without precipitating clinically significant GVHD. Our findings indicate that ipilimumab at 3 mg/kg is safe in this patient population. This is a relatively low dose compared with the doses used in phase III melanoma trials (10 mg/kg given every 2 weeks). Because dose might be important for optimal clinical effects, a higher dose and multiple administrations may provide a more potent and long-lasting GVM.

Previous studies have shown that T cells activated by cytokines such as IL-2 and interferon-γ have increased levels of both surface and intracellular CTLA-4 expression, where CTLA-4 plays an immunosuppressive role [41]. CTLA-4 blockade may not only affect Treg cell function, but also affect all activated T cells, leading to antitumor activity and autoimmune adverse events 42, 43. In 11 patients studied in our trial, intracellular CTLA-4 expression in CD4⁺CD25^lo/- T cells increased as early as day 1 after ipilimumab infusion and persisted for 2 months, whereas intracellular Foxp3 expression did not change significantly. At the same time, we observed the appearance of newly activated T cells. We speculate that the increased intracellular CTLA-4 levels might be associated with T cell activation.

Treatment for relapsed malignancy after allo-HSCT remains a major challenge. Methods to differentiate GVHD and GVM will be an intense focus of future research and clinical trials. Anti–CTLA-4 targeted therapy is one option to selectively activate the immune system, which may lead to augmented GVM activity. We are planning a subsquent study of multiple dosing of ipilimumab in this patient population when the antibody becomes available for clinical trials.

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Acknowledgments 

Financial disclosure: Edward D. Ball was a shareholder in Medaret at the time of the study.

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