Programmed death-1+ T cells and regulatory T cells are enriched in tumor-involved lymph nodes and associated with aggressive features in papillary thyroid cancer

Abstract

Context: Recurrent metastatic lymph node (LN) disease is common in patients with papillary thyroid cancer (PTC). Novel prognostic markers may be helpful in guiding a therapeutic approach. Our previous studies revealed that immune suppression is evident in PTC and associated with more severe disease.

Objective: To characterize the immune response to metastatic PTC, we assessed CD4(+) T cell polarization in LN. In addition, we investigated the role of programmed death-1 (PD-1) and T cell exhaustion.

Design: Uninvolved (UILN) and tumor-involved lymph nodes (TILN) were sampled ex vivo by fine-needle biopsy. T cell subsets were identified by flow cytometry. In parallel, archived TILN specimens were characterized by immunofluorescence.

Setting: The study was conducted at the University of Colorado Hospital.

Patients: Data were collected on 94 LN from 19 patients with PTC undergoing neck dissection.

Main outcome: T cell subset frequencies were compared in UILN and TILN and assessed for correlation with recurrent disease and extranodal invasion.

Results: Regulatory CD4(+) T cells (Treg) were enriched in TILN compared with UILN and further elevated in TILN from patients with recurrent disease. PD-1(+) T cells were present at high frequency in TILN and markedly enriched in TILN that showed evidence of extranodal invasion. In TILN, Treg frequency correlated with PD-1(+) T cell frequencies. Although PD-1(+) T cells produced interferon-γ, they failed to fully down-regulate CD27 and were not actively proliferating.

Conclusions: Increased Treg and PD-1(+) T cell frequencies in LN may be indicative of aggressive recurrent PTC. Future prospective studies are necessary to determine the prognostic and therapeutic value of these findings in PTC.

Fig. 1.

Analysis of T lymphocyte polarization in UILN and TILN by flow cytometry. Biopsies were obtained from UILN and TILN, and T cell subsets were analyzed by flow cytometry. A, A representative sample and the gating strategy is shown. B, CD4⁺ and CD8⁺ T cell subset frequencies were compared in UILN and TILN from patients with primary PTC. C, T cell subset frequencies were compared in TILN from patients with primary disease (PTC) and recurrent PTC (rPTC). D, Treg frequencies were compared in primary and recurrent patients in both TILN and UILN. Mean ± sem is shown. Values of P < 0.01 were considered significant.

Fig. 2.

PD-1⁺ T cell frequency in UILN and TILN. A, Biopsies were obtained from UILN and TILN, and PD-1⁺ T cell frequency was determined by flow cytometry. Cumulative graphs showing the percentage of PD-1⁺ cells in the general CD4⁺ and CD8⁺ T cell populations in UILN and TILN. Mean ± sem is shown. FoxP3⁺/CD4⁺ Treg and PD-1⁺/CD4⁺ (B) or PD-1⁺/CD8⁺ (C) T cells frequencies were compared in UILN and TILN. Values of P < 0.025 were considered significant.

Fig. 3.

PD-1⁺ T cell phenotype in UILN and TILN. A, Flow cytometry analysis of PD-1⁺ and PD-1⁻ CD4⁺ (top row) or CD8⁺ (bottom row) T cells. A representative TILN biopsy is shown. Matched PD-1⁻ and PD-1⁺ CD4 or CD8 T cells from each UILN or TILN were compared for expression of CD45RA (B) CD27 (C), and IFNγ (D). A subset of the patients contributed data for CD45RA (UILN, n = 9; TILN, n = 7) and CD27 (UILN, n = 37; TILN, n = 27).

Fig. 4.

Immunofluorescence analysis of PD-1 and Ki-67 in TILN. Lymph node tissue sections were stained for PD-1 (green) and Ki-67 (red) (A–C). Images were obtained at ×10 (A, LN4; and B, LN8) or ×60 (C, LN6) magnification. White arrows designated PTC metastasis (A and B). C, A single PD-1⁺Ki67⁺ lymphocyte is identified (white arrow) among PD-1⁺Ki-67⁻ lymphocytes. D, The frequency of PD-1⁺Ki67⁺ lymphocytes in the total PD-1⁺ population is shown for 12 TILN obtained from two patients with metastatic disease.

Fig. 5.

PD-1⁺ T cells, Treg, and extranodal invasion. PD-1⁺ T cell and Treg frequencies in each UILN and TILN were determined by flow cytometry. Archived TILN samples were analyzed for evidence of extranodal invasion by hematoxylin and eosin stain. Samples were grouped according to the absence or presence of invasion, and the frequency of FoxP3⁺CD4⁺ Treg (A), PD-1⁺CD4⁺ (B), or PD-1⁺CD8⁺ (C) T cells in each lymph node is shown. Mean ± sem is shown. Values of P < 0.017 were considered significant.