Combination immunotherapy induces distinct T-cell repertoire responses when administered to patients with different malignancies

Abstract

Background: CTLA-4 blockade with ipilimumab is Food and Drug Administration-approved for melanoma as a monotherapy and has been shown to modulate the circulating T-cell repertoire. We have previously reported clinical trials combining CTLA-4 blockade with granulocyte-macrophage colony-stimulating factor (GM-CSF) in metastatic melanoma patients and in metastatic castration resistant prostate cancer (mCRPC) patients. Here, we investigate the effect that cancer type has on circulating T cells in metastatic melanoma and mCRPC patients, treated with ipilimumab and GM-CSF.

Methods: We used next-generation sequencing of T-cell receptors (TCR) to compare the circulating T cells of melanoma and mCRPC patients receiving the same treatment with ipilimumab and GM-CSF by Wilcoxon rank sum test. Flow cytometry was utilized to investigate specific T-cell populations. TCR sequencing results were correlated with each T-cell subpopulation by Spearman's rank correlation coefficient. Of note, 14 metastatic melanoma patients had samples available for TCR sequencing and 21 had samples available for flow cytometry analysis; 37 mCRPC patients had samples available for sequencing of whom 22 have TCR data available at both timepoints; 20 of these patients had samples available for flow cytometry analysis and 16 had data available at both timepoints.

Results: While melanoma and mCRPC patients had similar pretreatment circulating T-cell counts, treatment induces greater expansion of circulating T cells in melanoma patients. Metastatic melanoma patients have a higher proportion of clones that increased more than fourfold after the treatment compared with mCRPC patients (18.9% vs 11.0%, p=0.017). Additionally, melanoma patients compared with mCRPC patients had a higher ratio of convergent frequency (1.22 vs 0.60, p=0.012). Decreases in clonality induced by treatment are associated with baseline CD8+ T-cell counts in both patient groups, but are more pronounced in the melanoma patients (r=-0.81, p<0.001 vs r=-0.59, p=0.02).

Trial registration numbers: NCT00064129; NCT01363206.

Keywords: immunology; medicine; oncology.

Figure 1

Modulation of different T-cell populations for mCRPC and melanoma patients with treatment. T-cell counts for specific populations were assessed by flow cytometry pretreatment after one cycle of treatmemt. (A) The count of CD4+ T cells. (B) The count of CD8+ T cells. (C) The count of CD25+ T cells. (D) The count of CD69+ T cells. (E) The count of CD127+ T cells. (F) The count of PD1+ T cells. (G) The count of CD25+CD69+CD127+PD1− T cells. (H) The count of CD25−CD69+CD127+PD1+ T cells. (I) The count of CD25−CD69+CD127+PD1− T cells. (J) The count of CD25−CD69−CD127+PD1+ populations. Each figure has two panels representing mCRPC (left) and metastatic melanoma (right). Each panel plots the population count at baseline and on-treatment with connected lines for each patient. The figures with * indicate that there is a significant difference in the ratio of on-treatment count and baseline count of each respective T-cell population between mCRPC and melanoma patients (p<0.05). mCRPC, metastatic castration resistant prostate cancer.

Figure 2

T-cell repertoire in melanoma patients undergo greater change with treatment compared with mCRPC patients. Changes in T-cell repertoire were assessed using NGS-based TCR sequencing. (A) The frequency of unique TCR for mCRPC and melanoma at baseline and with treatment are shown. Left and right scatter plots are from representative mCRPC and metastatic melanoma patients, respectively. Gray dots represent the clonotypes whose absolute log₂FC less than 2, where FC is defined as the ratio of on-treatment frequency versus baseline frequency. (B) Comparison of the proportion of T-cell clones that increased in frequency by more than fourfold after the treatment are shown for metastatic melanoma and mCRPC patients (18.9% vs 11.0%, respectively, p=0.017). (C) Morisita’s distance of clones present at both pretreatment and on-treatment is shown for metastatic melanoma and mCRPC patients (0.59 vs 0.72, respectively, p=0.077). (D) Ratio of TCR convergent frequency for metastatic melanoma and mCRPC patients (1.22 vs 0.60, p=0.012). mCRPC, metastatic castration resistant prostate cancer; NGS, next-generation sequencing; TCR, T-cell receptors.

Figure 3

Changes in TCR VJ gene usage induced with treatment in mCRPC and melanoma patients. VJ gene usage was assessed within pretreatment and on-treatment T cells. Each column represents an individual single VJ gene combination, and each row represents an individual patient with red bar and black bar in left side of the heat map representing melanoma and mCRPC patients, respectively. The color code represents the log₂(FC), where FC is the ratio of normalized gene usage at on-treatment versus baseline, with red to dark blue representing decreased to increased gene usage from baseline to on-treatment, respectively. mCRPC, metastatic castration resistant prostate cancer; TCR, T-cell receptors.

Figure 4

Greater reshuffling of high frequency T-cell clones in melanoma patients with treatment. (A) The change in rank of matched T-cell clones pretreatment and on-treatment for the top 100 T-cell clones at baseline is shown. (B) The SD of rank change in the top 100 clones in mCRPC and metastatic melanoma are shown. The left and right boxplots represent the SD of rank changes in the top 100 clones at baseline for mCRPC and metastatic melanoma patients, respectively. The SD of rank change is greater in melanoma patients than in mCRPC patients (p=0.045).

Figure 5

Associations between T-cell counts and T-cell repertoire. (A) CD8+ T-cell counts plotted against clonality at baseline for mCRPC patients where each point represents an individual patient and the line represents the linear correlation (r=0.71, p=0.03). (B) CD8+ counts plotted against clonality at baseline for metastatic melanoma patients (r=0.81, p<0.001). (C) CD8+ counts plotted against relative clonality for mCRPC patients (r=−0.59, p=0.022). (D) CD8+ counts plotted against relative clonality for metastatic melanoma patients (r=−0.82, p<0.001). mCRPC, metastatic castration resistant prostate cancer.