Long term impact of CTLA4 blockade immunotherapy on regulatory and effector immune responses in patients with melanoma

Abstract

Background: We previously reported early on-treatment significant modulation in circulating regulatory T cell (Treg), myeloid derived suppressor cells (MDSC) and antigen-specific type I CD4+ and CD8+ T cells that correlated with clinical outcome in regionally advanced melanoma patients treated with neoadjuvant ipilimumab. Here, we investigated the long term immunologic impact of CTLA4 blockade.

Methods: Patients were treated with ipilimumab given at 10 mg/kg IV every 3 weeks for 2 doses bracketing surgery. Blood specimens were collected at baseline and during treatment for up to 9 months. We tested immune responses at 3, 6, and 9 months utilizing multicolor flow cytometry. We compared frequencies of circulating Treg and MDSC on-study to baseline levels, as well as frequencies of CD4+ and CD8+ T cells specific to shared tumor-associated antigens (Gp-100, MART-1, NY-ESO-1).

Results: Levels of Treg significantly increased when measured at 6 weeks following ipilimumab but returned to baseline by 3 months, with no significant difference in Treg levels between relapsed and relapse-free groups at 3, 6 or 9 months. However, lower baseline levels of circulating Treg (CD4+CD25hi+CD39+) were significantly associated with better relapse free survival (RFS) (p = 0.04). Levels of circulating monocytic HLA-DR+/loCD14+ MDSC were lower at baseline in the relapse-free group and further decreased at 6 weeks, though the differences did not reach statistical significance including measurements at 3, 6 or 9 months. We detected evidence of type I (interferon-γ producing), activated (CD69+) CD4+ and CD8+ antigen-specific T cell immunity against cancer-testis (NY-ESO-1) as well as melanocytic lineage (MART-1, gp100) antigens in the absence of therapeutic vaccination. These responses were significantly boosted at 6 weeks and persisted at 3, 6 and 9 months following the initiation of ipilimumab.

Conclusions: Lower Treg levels at baseline are significantly associated with RFS and increased Treg frequency after CTLA4 blockade was only transient. Lower MDSC was also associated with RFS and MDSC levels were further decreased after ipilimumab. Tumor specific effector immune responses are boosted with CTLA4 blockade and tend to be durable. Trial registration ClinicalTrials.gov Identifier: NCT00972933.

Keywords: CTLA4; Ipilimumab; MDSC; Melanoma; Regulatory T cells; Tumor antigens.

Fig. 1

Neoadjuvant ipilimumab study design. Patients with locally and/or regionally advanced melanoma were enrolled and underwent excisional biopsy. They then received 2 cycles of ipilimumab 10 mg/kg every 21 days. A radical definitive surgery was then performed. Patients received 2 additional cycles of ipi 10 mg/kg every 21 days. Blood specimens (serum/peripheral blood monitoring) were collected at baseline, 6 weeks, 3, 6, and 9 months

Fig. 2

Frequencies of peripheral blood CD4+CD25^hi+CD39+ Treg at baseline and 3 months in patients who relapsed or were relapse-free at 9 months. a There was a significant difference in the percent of CD4+CD25hiCD39+ Treg at baseline between patients who had not relapsed at 9 months versus those who relapsed or died (p = 0.04). Those in the relapse group had significantly higher Treg levels. b By month 3, Treg in both groups tended to be higher compared to baseline, but to a greater degree in the relapse-free group. There was no significant difference in Treg levels at month 3 between the two groups (p = 0.266). Y axis shows the % of CD4⁺CD25^hi+ cells that express CD39

Fig. 3

Frequencies of CD4+CD25hiCD39+ Treg at baseline in healthy donors and patients. a Median levels of CD39+ Treg at baseline was lower in patients than in healthy donors, but the difference did not reach statistical significance (p = 0.144). b Median levels of CD39+ Treg at baseline was highest in healthy donors and lowest in non-relapsed patients, but the difference was not significant

Fig. 4

Frequencies of CD4+CD25hiFoxp3+ Treg and CD4+CD25hiCD39+ Treg from baseline through 9 months in patients who did not relapse or relapsed at 9 months. The solid blue line represents the median value of Treg for the relapse-free group, and the solid red line represents the median value for the relapsed group. a The levels of CD4+CD25hiFoxp3+ Treg increased at 6 weeks in both groups but returned to almost baseline levels starting 3 months. There were no significant differences in CD4+CD25hiFoxp3+ Treg between the two groups at baseline, 3, 6, or 9 months. b At 3 months, levels of CD4+CD25hiCD39+ Treg returned nearly to baseline. There were no significant differences in Treg levels between the two groups starting at 3 months

Fig. 5

Frequencies of circulating HLA-DR+lowCD14+ MDSC at baseline and 3 months. a Levels of circulating monocytic HLA-DR+lowCD14+ MDSC were lower at baseline in the relapse-free group. b Median levels increased in the non-relapsed groups at 3 months while staying the same in the relapsed group, but there were no significant differences in levels of MDSC at 3, 6, or 9 months between the 2 groups

Fig. 6

Forest Plots of Type I CD4 and CD8 Ag-specific T Cell Immunity (N = 27) at 6 weeks and at 3 months. Evidence of type I CD4+ and CD8+ antigen-specific T cell immunity against shared melanoma antigens, including lineage antigens (MART-1, gp-100) and cancer testis antigen (NY-ESO1), in the absence of prior vaccination. These responses were boosted after ipilimumab at a 6 weeks and b 3 months