Clonal expansion of CD8 T cells in the systemic circulation precedes development of ipilimumab-induced toxicities

Abstract

Immune checkpoint therapies, such as ipilimumab, induce dramatic antitumor responses in a subset of patients with advanced malignancies, but they may also induce inflammatory responses and toxicities termed immune-related adverse events (irAEs). These irAEs are often low grade and manageable, but severe irAEs may lead to prolonged hospitalizations or fatalities. Early intervention is necessary to minimize morbidities that occur with severe irAEs. However, correlative biomarkers are currently lacking. In a phase II clinical trial that treated 27 patients with metastatic prostate cancer, we aimed to test the safety and efficacy of androgen deprivation therapy plus ipilimumab. In this study, we observed grade 3 toxicities in >40% of treated patients, which led to early closure of the study. Because ipilimumab enhances T-cell responses, we hypothesized that increased clonal T-cell responses in the systemic circulation may contribute to irAEs. Sequencing of the T-cell receptor β-chains in purified T cells revealed clonal expansion of CD8 T cells, which occurred in blood samples collected before the onset of grade 2-3 irAEs. These initial results suggested that expansion of ≥55 CD8 T-cell clones preceded the development of severe irAEs. We further evaluated available blood samples from a second trial and determined that patients who experienced grade 2-3 irAEs also had expansion of ≥55 CD8 T-cell clones in blood samples collected before the onset of irAEs. We propose that CD8 T-cell clonal expansion may be a correlative biomarker to enable close monitoring and early intervention for patients receiving ipilimumab.

Keywords: CD8; T cells; ipilimumab; prostate cancer; toxicities.

Fig. 1.

Clinical trial schema and clinical responses. (A) Clinical trial treatment [ipilimumab (IPI) plus finite ADT] and blood draw schemata. (B) Time to PSA progression from treatment initiation based on PCWG2 criteria. The y axis numbers each individual patient based on initiation of treatment with ipilimumab. Each bar represents an individual patient. The arrowhead depicts that the patient’s clinical response is ongoing. (C) Radiographic responses for patient 1. MRI of the pelvis of patient 1 at baseline and posttreatment. (a) A 2.0- × 2.4-cm infiltrating mass in the right peripheral zone of a 6.5- × 5.1- × 5.6-cm prostate. (b) A 3.4- × 1.5-cm metastasis involving the right inferior pubic ramus. (c) Resolution of the prostatic mass. (d) Resolution of the bony metastasis following treatment with ADT plus ipilimumab.

Fig. S1.

Flow cytometry-based peripheral blood candidate biomarkers. (A) CD4 T-cell ICOS expression as a pharmacodynamic marker of ipilimumab treatment. Flow cytometry analysis of ICOS expression of CD4 T cells (as a percentage of CD3CD4 T cells) before each dose of ipilimumab [IPI; baseline (pre-IPI dose 1) and following each dose] PBMCs. Each symbol represents an individual patient. The error bars represent mean and SD. (B) Baseline frequency of costimulatory markers in CD3 (Top) and CD4 (Bottom) T cells in the peripheral blood based on PSA responses. Flow cytometry analysis of baseline (pre-IPI treatment) PBMCs for expression of CTLA-4, OX-40, and PD-1 (as a percentage of CD3 and CD4 T cells) and CTLA-4:PD-1. Each symbol represents an individual patient. The error bars represent mean and SD.

Fig. 2.

Evaluation of immunological biomarkers that correlate with irAEs. (A) Scatter plots of CD4 and CD8 TCR clone frequencies within sorted T-cell populations from post-IPI dose 1 or 2 samples vs. pre-IPI (baseline) treatment samples for patients with no toxicity at the time of sample collection (patients 17 and 28), a patient experiencing grade 3 transaminitis (patient 3), and a patient experiencing grade 3 diarrhea (patient 11). (B) Comparison between patients who experienced grade 0–1 irAEs vs. grade 2–3 irAEs. The number of expanded of T-cell clones was determined in samples collected just before a grade 2–3 irAE relative to pre-IPI treatment samples vs. patients with grade 0–1 irAEs in both clinical trials. For patients with a grade 0 toxicity, we used post-IPI dose 1 samples to identify expanded clones.

Fig. S2.

Enrichment of CD4 and CD8 T cells from PBMCs and immunological biomarkers. (A) (Top) CD4 and CD8 T-cell expression in PBMCs based on flow cytometric analysis. Following purification of CD4 T cells (negative selection) and CD8 T cells (positive selection) from PBMCs, flow cytometric analysis was used to determine the purity of the CD4 (Bottom Right) and CD8 (Bottom Left) T-cell populations. In this patient, the CD4 population is enriched from 43.7% to 98.3% purity, and the CD8 population is enriched from 37.7% to 95.9%. (B) Baseline CD4 and CD8 T-cell clonality from pre-IPI (baseline) treatment samples were used to determine T-cell clonality and to make comparisons between patients who experienced PSA progression greater than 16 mo (clinical benefit) vs. less than 8 mo (no clinical benefit). (C) Baseline CD4 and CD8 T-cell clonality from pre-IPI (baseline) treatment samples was used to determine T-cell clonality and to make comparisons between patients who experienced grade 0–1 irAEs vs. grade 2–3 irAEs. (D) Comparison between patients who experienced PSA progression greater than 16 mo (clinical benefit) vs. less than 8 mo (no clinical benefit). The number of expanded T-cell clones was determined from post-IPI dose 1 vs. baseline samples.

Fig. 3.

CD8 clonal expansion precedes grade 2–3 irAEs. (A) The ROC curve of expanded CD8 clones. (B) Comparison between pooled patients from two clinical trials who experienced grade 0–1 irAEs vs. grade 2–3 irAEs. The number of expanded T-cell clones was determined in samples collected just before a grade 2–3 irAE relative to pre-IPI treatment samples vs. patients with grade 0–1 irAEs in both clinical trials. For patients with a grade 0 toxicity, we used post-IPI dose 1 samples to identify expanded clones.