C reactive protein impairs adaptive immunity in immune cells of patients with melanoma

Abstract

Background: High C reactive protein (CRP) levels have been reported to be associated with a poor clinical outcome in a number of malignancies and with programmed cell death protein 1 immune checkpoint blockade in patients with advanced cancer. Little is known about the direct effects of CRP on adaptive immunity in cancer. Therefore, we investigated how CRP impacted the function of T cells and dendritic cells (DCs) from patients with melanoma.

Methods: The effects of CRP on proliferation, function, gene expression and phenotype of patient T cells and DCs, and expansion of MART-1 antigen-specific T cells were analyzed by multicolor flow cytometry and RNA-seq. Additionally, serum CRP levels at baseline from patients with metastatic melanoma treated on the Checkmate-064 clinical trial were assessed by a Luminex assay.

Results: In vitro, CRP inhibited proliferation, activation-associated phenotypes and the effector function of activated CD4+ and CD8+ T cells from patients with melanoma. CRP-treated T cells expressed high levels of interleukin-1β, which is known to enhance CRP production from the liver. CRP also suppressed formation of the immune synapse and inhibited early events in T-cell receptor engagement. In addition, CRP downregulated the expression of costimulatory molecules on mature DCs and suppressed expansion of MART-1-specific CD8+ T cells in a dose-dependent manner by impacting on both T cells and antigen-presenting cells. High-serum CRP levels at baseline were significantly associated with a shorter survival in both nivolumab-treated and ipilimumab-treated patients.

Conclusions: These findings suggest that high levels of CRP induce an immunosuppressive milieu in melanoma and support the blockade of CRP as a therapeutic strategy to enhance immune checkpoint therapies in cancer.

Trial registration number: NCT01783938 and NCT02983006.

Keywords: immunology; medicine; oncology.

Conflict of interest statement

Competing interests: JW: honoraria and travel from BMS, Merck, GSK, Genentech, AstraZeneca, Pfizer, CytoMx, EMD Serono, Incyte. Stock in Biond, Altor, Protean, CytoMx, Celldex, Sellas. Research funding from NextCure. All other clinical research funding to my institution, not me. Named on a patent for a PD-1 biomarker by Biodesix not used in this work. Named on a CTLA-4 biomarker patent by Moffitt Cancer Center not used in this work. Named on a patent for the use of 41-BB antibody for tumor infiltrating lymphocyte growth by Moffitt Cancer Center not used in this work.

© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1

C reactive protein (CRP) affects the proliferation on activated T cells. CD3+ T cells were isolated by negative magnetic separation from baseline patient peripheral blood mononuclear cells (PBMC), and activated with CD3/CD28 antibody for 72 hours in the presence of different CRP concentration. (A) Proliferation analysis of CellTrace Violet-labeled CD3+ T cells stimulated with anti-CD3 and CD28 monoclonal antibodies (mAbs) for 72 hours. The cell ratio and CRP+ population of CD8+ cells in each generation (G1 to over G4) were detected. *P

Figure 2

C reactive protein (CRP) affects the function of activated T cells. Expression of checkpoint, stimulatory and cytolytic proteins by flow cytometry on activated CD8 (A) and CD4 (B) T cells treated with CRP at 10, 20 and 40 µg/mL as a heat map. Representative data from three patients. (C) CD3+ T cells isolated from baseline peripheral blood mononuclear cells (PBMC) samples of patients with melanoma prior to any treatment were cultured for 72 hours with or without CRP at doses of 10, 40 and 100 µg/mL, and subsequently stimulated for 4 hours with phorbol 12-myristate 13-acetate (PMA)/ionomycin for analysis of intracellular cytokine production (interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2) and granzyme B. Representative data from five patients. Error bars are +SEM for triplicate samples assessed. **P

Figure 3

C reactive protein (CRP) inhibits T-cell receptor (TCR) signaling pathway in T cells. (A) Expression of phosphorylated LCK (pY505), ZAP70(pY319)/Syk(pY352), LAT(pY717), ERK(pT202/pY204) and AKT(pS473) on CD3+ T cells pretreated with CRP at 10, 40 and 100 µg/mL for 72 hours and concomitantly stimulated with H2O2 are from one representative of five similar patients. *P<0.05, **p<0.01, ***p<0.001 relative to cells with no treatment. (B) Representative data from three patients of calcium influx on T cells. CD3+ T cells treated with CRP for 24 hours at 10–100 μg/mL were activated with phorbol 12-myristate 13-acetate (PMA) and ionomycin. (C and D) Anti-CD3/CD28 monoclonal antibodies (mAbs) Dynabeads recruit lipid rafts of CD3+ cells to form immunological synapses. CRP (40 µg/mL) treated CD3+ T cells stained with anti-CRP antibody (green) were significantly less likely to recruit lipid rafts to form the immunological synapse at the bead-T cell interface denoted by Phallodin staining (red) that control treated CD3+ T cells. Representative tiled image and high-magnification images from one of three patients.

Figure 4

C reactive protein (CRP) inhibits function, costimulatory and checkpoint proteins on dendritic cells (DCs). Frequency of mature (A) and immature (B) DCs treated with CRP at 10 and 40 μg/mL for 48 hours expressed costimulatory markers (CD40, CD80, CD83, CD86, PD-L1, HLA-ABC, HLA-DR and beta-2 microglobulin). Representative data are from one of five patients. Error bars are +SEM for triplicate samples assessed. **P

Figure 5

C reactive protein (CRP) inhibits the expansion of antigen-specific T cells. (A) MART-1-specific CD8 T-cell expansion. CD8+ T cells isolated from HLA-A*0201 patients with melanoma were stimulated by γ-irradiated, MART-126-35 peptide-pulsed peripheral blood mononuclear cells (PBMCs) (antigen-presenting cells (APCs)) for 7 days in presence of different CRP concentration (0, 10, 40 µg/mL). (B and C) Ki67 expression and proliferation by CFSE-labeled Melan-A-specific CD8+ T cells and MART-1 tetramer-positive T cells stimulated by γ-irradiated MART-126-35 peptide pulsed APC. (D) Scheme for assessing whether CRP-impaired generation of Melan-A-positive CD8+ T cells was due to its effect on T cells and/or on mature dendritic cells (mDCs). (E) Purified CD8+ T cells and mDCs separately before co-culture were treated with CRP for 48 hours, and co-cultured with MART-1 peptide-pulsed mDCs. Results shown are representative of three patients assessed over three independent experiments. (F) Volcano plot for the differential expression of total genes. The y-axis corresponds to negative log10 transformed adjusted p value, and the x-axis displays the log2 fold change gene expression value. The red and blue dots represent the significantly differential expressed transcripts (adjusted p value < 0.05); the gray dots represent the transcripts whose expression levels did not reach statistical significance (adjusted p value > 0.05). (G) Expression heat map of genes associated with cytokine-cytokine receptor interaction pathway, and osteoclast differentiation pathway. Error bars are +SEM for triplicate samples assessed. **P<0.05, **p<0.01, ***p<0.001 relative to cells with no CRP treatment.

Figure 6

Serum C reactive protein (CRP) levels in treatment-naïve patients receiving nivolumab (cohort A) or ipilimumab (cohort B) from the Checkmate-064 trial. (A) Consolidated Standards of Reporting Trials diagram for the study. Among 140 patients enrolled in the Checkmate-064 trial, 95 serum samples before administration of study drug were obtained for CRP analysis (29 from cohort A, 66 from cohort B). (B) Kaplan-Meier plot of the relation of overall survival (OS) to CRP levels before treatment. Cut-off point was at the median (15.48 µg/mL). The red curve represents survival for patients below the median, and the blue curve shows survival for those patients above the median. The survival probabilities were estimated using the Kaplan-Meier method, where differences in the variables were calculated using the log-rank test.