PD-1 Blockade Expands Intratumoral Memory T Cells - PubMed (original) (raw)

Clinical Trial

doi: 10.1158/2326-6066.CIR-15-0210. Epub 2016 Jan 19.

Daniel Sanghoon Shin 2, Jesse Zaretsky 2, Juliet Frederiksen 3, Andrew Cornish 4, Earl Avramis 2, Elizabeth Seja 2, Christine Kivork 2, Janet Siebert 5, Paula Kaplan-Lefko 2, Xiaoyan Wang 6, Bartosz Chmielowski 2, John A Glaspy 2, Paul C Tumeh 7, Thinle Chodon 8, Dana Pe'er 4, Begoña Comin-Anduix 9

Affiliations

Clinical Trial

PD-1 Blockade Expands Intratumoral Memory T Cells

Antoni Ribas et al. Cancer Immunol Res. 2016 Mar.

Abstract

Tumor responses to programmed cell death protein 1 (PD-1) blockade therapy are mediated by T cells, which we characterized in 102 tumor biopsies obtained from 53 patients treated with pembrolizumab, an antibody to PD-1. Biopsies were dissociated, and single-cell infiltrates were analyzed by multicolor flow cytometry using two computational approaches to resolve the leukocyte phenotypes at the single-cell level. There was a statistically significant increase in the frequency of T cells in patients who responded to therapy. The frequency of intratumoral B cells and monocytic myeloid-derived suppressor cells significantly increased in patients' biopsies taken on treatment. The percentage of cells with a regulatory T-cell phenotype, monocytes, and natural killer cells did not change while on PD-1 blockade therapy. CD8(+) memory T cells were the most prominent phenotype that expanded intratumorally on therapy. However, the frequency of CD4(+) effector memory T cells significantly decreased on treatment, whereas CD4(+) effector T cells significantly increased in nonresponding tumors on therapy. In peripheral blood, an unusual population of blood cells expressing CD56 was detected in two patients with regressing melanoma. In conclusion, PD-1 blockade increases the frequency of T cells, B cells, and myeloid-derived suppressor cells in tumors, with the CD8(+) effector memory T-cell subset being the major T-cell phenotype expanded in patients with a response to therapy.

©2016 American Association for Cancer Research.

PubMed Disclaimer

Conflict of interest statement

Competing interests: Competing interests: Dr. A. Ribas has served as consultant for Merck, with the honoraria paid to UCLA. B. Chmielowski has received honoraria from BMS, Genentech, and Prometheus, and has served as a consultant for or on the advisory boards of Genentech, Amgen, Lilly, Astellas, Merck, and BMS.

Figures

Figure 1

Figure 1. Changes in leukocyte subpopulations on PD-1 blockade therapy in tumor samples

**(A)**Frequency of leukocytes (CD45+) before (B, n = 27) and on (n = 24) anti–PD-1 therapy. (B) Among leukocytes, percentage of T cells (CD3+; * P = 0.02), monocytes (CD14+; P = 0.476), NK (CD56+; P = 0.47) and B cells (CD19/20+; * P = 0.04). (n=29 before therapy; n=25, on therapy). (C) Proportion of the ratio for CD8/CD4 cells in paired samples (n= 22 pairs; P = 0.0542; Wilcoxon test). (D) Changes in the percentage of T reg. (n=42 before, n=35 on therapy; P = 0.54). (E) Changes in the percentage of moMDSC.(n=27 before, n=25 on therapy; *, P = 0.04). Mean and ± SD are provided. Solid circles represent responders; open circles represent non-responders. B = before treatment; P = on anti–PD-1 therapy; n = number of biopsies analyzed.

Figure 2

Figure 2. Phenotype analysis of T cells from baseline tumors

The following CD3+ T cells markers were plotted prior to any treatment. In order: CD45RA, n = 62; CD45RO, n = 58; CD27, n = 62; CD28, n = 19; CD127, n = 19; CCR7, n = 62; CD62L; n = 62; CD57, n = 43; CD95, n = 62; and PD-1, n = 44. All the markers expressed in percentages. Mean and ± SD are provided. n = number of biopsies analyzed. Solid circles represent responders; open circles represent non-responders.

Figure 3

Figure 3. CD8+ T cell subsets with significant differences in biopsies on PD-1 blockade therapy

(A) Dendogram illustrating the frequency of CD8+ T cell subsets that present lower P value (P ≤ 0.05) with higher 95% confidence interval (95% CI) using CytoAnalysis program. The program presented an increase of the mean difference (MD) and the 95% CI of the baseline and post-dose samples as a negative value. (n) describes how many patients of the 18 paired TILs present with that particular phenotype. (B) Analysis of the percentage of CD8+CD4-CD45RO+ phenotype (*P = 0.02), displaying a higher percentage the responders (**P = 0.002). (C) Analysis of the percentage of the more extended combination of phenotype markers CD8+CD4-CD45RO+CCR7-CD27-CD57- (*P = 0.01), being augmented in responding patients (**P = 0.006) (right plot). The on-going treatment (red circle) was arranged by responder and non-responders tumors (right plot). Mean and ± SD are provided. B = before treatment; P = on anti–PD-1 therapy; n = number of biopsies

Figure 4

Figure 4. CD4+ T cell subsets with significant differences in biopsies on PD-1 blockade therapy

(A) Dendogram illustrating all CD4+ T cells that present with a lower P value (P ≤ 0.05) with higher 95%CI using CytoAnalysis program. The program presents as positive values a decrease of the mean difference (MD) and the 95% CI of the baseline and post-dose samples as a positive value. (n) describes how many patients of the 18 paired TILs show that particular phenotype. (B) The percentage of a phenotype of T effector CD8-CD4+CD45RO+CD57+ T cells (*P = 0.05) plus two related more extended phenotypes, CD8-CD4+CD45RO+CCR7-CD57+ (*P = 0.003) and CD8−CD4+CD45RO+CCR7-CD62L-CD57+ (*P = 0.05). The on-going treatment (red circle) was compared between responder and non-responders tumors (right plot), being the P value: CD8-CD4+CD45RO+CD57+: * P = 0.024; CD8-CD4+CD45RO+CCR7-CD57+: *P = 0.022; and CD8-CD4+CD45RO+CCR7-CD62L-CD57+ (p>0.05)). B = before treatment; P = on anti–PD-1 therapy. Mean and ± SD are provided. Solid circles represent responders; open circles represent non-responders (n = 46 baseline; n = 37 treated).

Figure 5

Figure 5. Comparison of CD8+ T memory phenotypes in paired biopsies from patients with a response to anti–PD-1 therapy

(A) Paired longitudinal analysis of the frequency of CD8+CD4−CD45RO+ at baseline and on anti–PD-1 treatment by exhaustive expansion software. PXX = patient number. B = baseline. DXXX = day of blood draw from the start of anti–PD-1 treatment. n= 9 patients. (B) Visualization of the CD45RO T cells from the suprarenal biopsy from patient #47 nineteen days on therapy. Contour plots of the viSNE maps for double positive for CD3 and live gated cells (excluding CD16 and CD19). The contour plots show cell density in each region of the map. Each dot in the viSNE map depicts the location of an individual cell and its color represents the expression of its immune cell marker. CD3+ T cells (left column) contours plots before (blue), and on anti–PD-1 blockade therapy (red). The expression of CD8+ T (in red) and CD4+ T (in blue) cells inside the CD3 T cell contour plot are shown in the middle column. On the right column, CD45RO (in red) is shown. Red color means high, orange means medium and blue represents low expression of the marker on the cell surface.

Figure 6

Figure 6. Analysis of peripheral blood samples at baseline and on anti–PD1 therapy

(A) Dot plots depicting the percentage of leukocytes (CD45+) on the left, the percentage of T (CD3+), monocytes (CD14+), B (CD19+/CD20+) and NK (CD56+) in the center, and the ratio CD8/CD4 on the right (B, n = 9; P n = 13). (B) Dot plots of suppressor cells. Tregs on the left, and moMDSC on the right. (C) Dot plots of the T cell subset, CD45RA, CD45RO, CD27, CCR7, CD62L, CD57, CD95 (B, n = 9; P n = 14), and PD-1 only at baseline (n = 9). Mean and ± SD are provided. B = Before treatment; P = on anti–PD-1 therapy; n = number of biopsies analyzed. Solid circles, responders; open circles, nonresponders (D, E) viSNE analysis from blood from two patients who responded to anti–PD-1 (patients #4 and #45) identified differences between the baseline and on treatment samples. (D) The viSNE map of the baseline (blue) and on treatment (green) sample showed the presence of a distinct population (events within the red circle). (E) Histogram representation of the population of interest (red) identified from the viSNE plot at baseline (blue) and on treatment (green). The CD19, CD56, CD14, CD3, CD4, CD127, CD25, and HLA-DR channels of the baseline and on treatment samples were chosen for analysis by viSNE. The marker expression is shown on the x-axis and the density of cells is shown on the y-axis.

Similar articles

Cited by

References

    1. Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer. 2012;12(4):252–64. - PMC - PubMed
    1. Ishida Y, Agata Y, Shibahara K, Honjo T. Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death. EMBO J. 1992;11(11):3887–95. - PMC - PubMed
    1. Ahmadzadeh M, Johnson LA, Heemskerk B, Wunderlich JR, Dudley ME, White DE, et al. Tumor antigen-specific CD8 T cells infiltrating the tumor express high levels of PD-1 and are functionally impaired. Blood. 2009;114(8):1537–44. - PMC - PubMed
    1. Sheppard KA, Fitz LJ, Lee JM, Benander C, George JA, Wooters J, et al. PD-1 inhibits T-cell receptor induced phosphorylation of the ZAP70/CD3zeta signalosome and downstream signaling to PKCtheta. FEBS Lett. 2004;574(1-3):37–41. - PubMed
    1. Okazaki T, Maeda A, Nishimura H, Kurosaki T, Honjo T. PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting src homology 2-domain-containing tyrosine phosphatase 2 to phosphotyrosine. Proc Natl Acad Sci U S A. 2001;98(24):13866–71. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources