MHC class I loss is a frequent mechanism of immune escape in papillary thyroid cancer that is reversed by interferon and selumetinib treatment in vitro

Abstract

Purpose: To evaluate MHC class I expression on papillary thyroid cancer (PTC) and analyze changes in MHC expression and associated immune activation with current and experimental treatments for thyroid cancer using in vitro PTC cell lines.

Experimental design: MHC class I expression and assessment of tumor-infiltrating leukocyte populations were evaluated by immunohistochemistry. PTC cell lines were analyzed for HLA-ABC expression by flow cytometry following tyrosine kinase inhibitor, IFNα or IFNγ, or radiation treatment. Functional changes in antigenicity were assessed by coculture of allogeneic donor peripheral blood leukocytes (PBL) with pretreated or untreated PTC cell lines and measurement of T-cell activation and cytokine production.

Results: Both MHC class I and β2-microglobulin expression was reduced or absent in 76% of PTC specimens and was associated with reduced tumor-infiltrating immune cells, including effector (CD3(+), CD8(+), CD16(+)) and suppressor (FoxP3(+)) populations. Treatment of PTC cell lines with the MEK1/2 inhibitor selumetinib or IFN increased HLA-ABC expression. This phenotypic change was associated with increased T-cell activation (%CD25(+) of CD3(+)) and IL2 production by PBL cocultured with treated PTC cell lines. Additive effects were seen with combination selumetinib and IFN treatment.

Conclusions: MHC class I expression loss is frequent in human PTC specimens and represents a significant mechanism of immune escape. Increased antigenicity following selumetinib and IFN treatment warrants further study for immunotherapy of progressive PTC.

Figure 1

Frequent loss of MHC Class I expression in PTC (A) Bar graph showing the number of specimens with intact, reduced, or absent expression of HLA-ABC or beta 2 microglobulin (β2m). (B) IHC staining of representative PTC tumor specimens for HLA-ABC and β2m demonstrating (from left to right) intact and absent expression in PTC specimens, lymph node control, and intact expression in normal thyroid follicular cells (original magnification, 400×). Black arrow indicates membrane localization of staining for HLA-ABC consistent with intact expression.

Figure 2. Correlation of tumor HLA-ABC expression with the frequency of tumor-infiltrating leukocytes

(A) Mean (+SEM) CD3⁺ and CD8⁺ cells/hpf infiltration in PTC with intact versus reduced/absent HLA-ABC and beta 2 microglobulin (β2m). Statistically significant differences between groups is indicated by * p<0.05, ** p<0.01. (B) Linear regression analysis between mean CD3⁺ cells/hpf infiltration and increasing HLA-ABC expression score demonstrates a significant positive correlation (r²=0.29, p=0.0011). (C) TIL infiltration in PTC tumors with intact versus reduced/absent MHC class I expression, demonstrating higher mean CD16⁺, FoxP3⁺, CD68⁺, and CD163⁺ cells/hpf + SEM. Differences between groups were not statistically significant.

Figure 3. Select tyrosine kinase inhibitors increase the antigenicity of PTC cell lines

(A) The effect of in vitro treatment of PTC cell lines with kinase inhibitors sunitinib, sorafenib, and selumetinibon HLA-ABC expression as measured by flow cytometry. Representative flow cytometry histograms for PTC cell lines (BCPAP, K-1, and TPC-1) stained with fluorescence-labeled monoclonal antibodies after treatment with sunitinib, selumetinib, or vehicle/media control. (B, C) Expression of immunosuppressive ligands PD-L1 and HLA-G by PTC cell lines following sunitinib or selumetinib treatment. For all graphs in A-C, mean +SEM is shown, with statistically significant difference from vehicle control indicated by * p<0.05, ** p<0.01, *** p<0.001. (D) T cell activation measured as the CD25⁺ fraction of CD3⁺ T cells in PBL co-cultured with PTC after pre-treatment with drug or vehicle control. (E) T cell activation as measured by IL-2 production in PBL-PTC cell line co-cultures after PTC were pre-treated with drug or vehicle control. For D and E, data shown are mean (n=4) +SEM, with significant difference from vehicle control-treated PTC indicated by * p<0.05, ** p<0.01. HLA-G = human leukocyte antigen G; PD-L1 = programmed death ligand 1.

Figure 4. IFN produces significant up-regulation of HLA-ABC in PTC corresponding to increased antigenicity

(A) The effect of IFNα or IFNγ in vitro treatment of PTC cell lines on HLA-ABC expression measured by flow cytometry. Data shown are mean +SEM and significant differences from vehicle control are indicated by * p<0.05, ** p<0.01, *** p<0.001. Representative flow cytometry histograms for HLA-ABC expression are shown to the right. (B) T cell activation CD25⁺ fraction of CD3⁺ T cells or (C) IL-2 production in PBL-PTC cell line co-cultures after PTC were pre-treated with interferon or vehicle control. For B and C, data shown are mean (n=4) +SEM, with significant differences from vehicle control-treated PTC indicated by * p<0.05, ** p<0.01, *** p<0.001.

Figure 5. Combination selumetinib and IFN treatment of PTC cell lines produces additive increases in HLA-ABC expression and antigenicity

(A) The effect of selumetinib and IFNγ or IFNα treatment on PTC HLA-ABC expression. Data shown are mean +SEM, with statistically significant differences from vehicle control indicated by * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Representative flow cytometry histograms for HLA-ABC expression are shown to the right of the respective graph. (B) T cell activation measured as the fraction CD25⁺ of CD3⁺ T cells or (C) IL-2 production in PBL-PTC co-cultures after PTC were pre-treated with interferon, selumetinib, or combination therapy. For B and C, data shown are mean (n=4) +SEM, with significant differences from single reagent therapy indicated by * p<0.05, ** p<0.01, *** p<0.001.