CTLA-4⁺ Regulatory T Cells Increased in Cetuximab-Treated Head and Neck Cancer Patients Suppress NK Cell Cytotoxicity and Correlate with Poor Prognosis

Cetuximab monotherapy increases CD25⁺, CTLA-4⁺, CD39⁺, and LAP⁺ (membrane bound TGFβ⁺) cells mainly on intratumoral Foxp3⁺ Treg isolated from 18 patients with HNSCC. Flow cytometric analysis of circulating and intratumoral CD4⁺CD25^hiFoxp3⁺ Treg isolated from a representative HNSCC patient treated with single-agent cetuximab therapy on a prospective phase II clinical trial (UPCI #08-013). PBL and TIL were respectively isolated from blood and tumors of patients with HNSCC pre- and post-single-agent cetuximab therapy. Percentages of CD25⁺ (A), CTLA-4⁺ (C), CD39⁺ (E), and LAP⁺ (G) cells in CD4⁺Foxp3⁺ Treg were compared pre- and post-single-agent cetuximab therapy for a series of patients with HNSCC. Representative flow cytometric analysis of CD25 (B), CTLA-4 (D), CD39 (F), and LAP (H) on Foxp3⁺ Treg of CD4⁺ PBL and TIL isolated from a HNSCC patient (pre- and post-cetuximab treatment). The numbers represent the percentages of CD25⁺, CTLA-4⁺, CD39⁺, LAP⁺, and Foxp3⁺ cells in CD4⁺ T cells.

Treatment with cetuximab combined with TCR triggering induces CTLA-4⁺ Treg expansion. JHU029 cell line and NK cells (1:1 ratio) were cocultured in the presence of cetuximab or human IgG1 at the upper chamber of transwell plate, whereas at the lower chamber, purified monocytes and CFSE-labeled CD4⁺ T cells (1:2 ratio) were cultured with TGFβ1 in the presence or absence of anti-CD3 antibody. Similar results were seen of lower magnitude when TGFβ1 was omitted from the cultures. Four days after incubation, the frequency (A and B) and proliferation (C and D) of CTLA-4⁺Foxp3⁺ Treg were assessed by flow cytometry using the PerCP-Cy5.5 dye. Representative flow cytometric analysis of CTLA-4⁺Foxp3⁺ Treg (A) and their proliferation by CFSE dilution (C) are shown for each condition and their frequency was statistically compared, respectively (B and D).

Cetuximab monotherapy differentially activates circulating and intratumoral NK cells. PBL and TIL were isolated from patients with HNSCC before and after single-agent cetuximab therapy to analyze CD3⁻CD56⁺ NK cells for their cytolytic activity–related molecules (granzyme B, perforin, and CD16), degranulation marker (CD107a), and activation molecule (CD137). Percentages of CD3⁻CD56⁺ NK cells (A), CD16⁺(B), granzyme B⁺(C), perforin⁺(D), CD107a⁺(E), CD137⁺ (F) cells of circulating and intratumoral CD3⁻CD56⁺ NK cells were compared in pre- and post-single-agent cetuximab therapy for patients with HNSCC (n = 17).

Cetuximab monotherapy increases the frequency of CTLA-4⁺ Treg only in nonresponder group. The frequency of CTLA-4⁺ Treg in CD4⁺ PBL (A) and TIL (B) was measured using flow cytometry and compared in pre- and post-single-agent cetuximab monotherapy for 5 responders and 13 nonresponders respectively.