Thyroid cancer (TC) is the fifth most common cancer in women in the USA, with an estimated 52,890 new TC cases nationwide in 2020 (1). The incidence of TC has been increasing in recent decades, and showing a trend of rejuvenation (2–7). TC also has the highest rate of growth among diagnosed tumors in the USA (8). Papillary thyroid carcinoma (PTC) belongs to differentiated thyroid carcinoma (DTC) and accounts for approximately 80–85% of all TCs (2, 9). Although the vast majority of PTCs can be cured by surgery and ¹³¹I treatment, 10% of patients still die from poorly differentiated and advanced tumors (10). A study with a median follow-up period of 27 years also showed that 28% of PTC patients relapsed and 9% died of PTC (11). The reason is partly because the interaction and collective effect of tumors with the surrounding immune microenvironment during the occurrence and development of the tumor promotes immune tolerance and then progresses to immune escape, resulting in the body’s inability to completely clear the tumor.

The immune editing hypothesis was proposed by Dunn in 2002 and divided the interaction between the immune system and the tumor into three stages: (1) elimination: tumor cells are cleared by the immune system before a clinical diagnosis is established. This stage is also called immune surveillance; (2) equilibrium: tumor cells with less immunogenicity gradually replace tumor cells with more immunogenicity; and (3) escape: tumor cells eventually escape the body’s immune surveillance (12). According to this hypothesis, the immune editing of tumors also sculpts the tumor’s immune phenotype while clearing the tumor, enabling the host to generate immune activity against the tumor. When the tumor is clinically diagnosed, it can not only tolerate the immune response but also manipulate the immune system to promote tumor progression. Poschke et al. (13) believe that tumors have two different methods of immune escape: camouflage and sabotage. Camouflage refers to the malformation and loss of the major histocompatibility complex class I (MHC-I) molecules on the surfaces of tumors, allowing tumors to escape the detection of the immune system. Sabotage refers to the ability of some tumors, including PTC, to manipulate part of the immune system to fight against the body’s immune response to protect themselves. In this process, tumors attract and even mediate some immune cells, such as tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), Tregs, and mast cells (MCs). In general, MDSCs, Tregs, and MCs respond to uncontrolled inflammatory reactions in the body, but in tumor progression, they have created an immune microenvironment that allows this process. Existing evidence shows that Tregs, neutrophils, dendritic cells (DCs), MCs, and TAMs play a tumorigenic role in the PTC tumor microenvironment (TME), while CD8+ T cells, B cells, and NK cells play a protective role (14–16). However, the infiltration and role of some major immune cells in PTC are still controversial. Studies on nonprimary immune cells are scarce, and systematic research on the PTC immune microenvironment remains to be conducted. Therefore, accurate assessment of the specific pattern of immune cells in PTC, including not only its phenotype but also its function, is essential for subsequent immune research and new diagnostic and therapeutic strategies.

The purpose of this study was to systematically understand immune cell infiltration in the PTC microenvironment and to summarize the changes in the immune microenvironment during the occurrence and development of PTC. Our research found that M2 macrophages, Tregs, monocytes, neutrophils, DCs, MCs, and M0 macrophages play a tumor-promoting role, while M1 macrophages, CD8+ T cells, B cells, NK cells, and T follicular helper (T_FH) cells play an antitumor role. During the occurrence and development of PTC, the overall immune level increased, and the abundance and proportion of tumor-promoting immune cells increased significantly. Our research will help researchers systematically understand PTC immune cell infiltration and provide a reference for subsequent basic and clinical research related to PTC immunity.

Our findings showed that during the occurrence and development of PTC, the immune system is enhanced; moreover, M2 macrophages, Tregs, monocytes, neutrophils, DCs, MCs, and M0 macrophages play a tumor-promoting role, and their abundances and proportions are significantly increased. M1 macrophages, CD8+ T cells, B cells, NK cells, and T_FH cells (eosinophils, γδ T cells, and Th17 cells with weak supporting evidence) exert an antitumor effect, and the proportions decreased.

PTC is subdivided into “inflammatory” tumors (71). Extensive evidence shows that autoimmunity and inflammation are risk factors for TC (72). Increasing evidence also suggests that cancer-related inflammation may be a useful target for new diagnostic and therapeutic strategies in TCs (73). Therefore, to evaluate the specific pattern of immune cells involved in PTC, including not only its phenotype but also its function, it is essential to understand the immunological characteristics of PTC. Many major advances have been made in the study of immune infiltration in PTC, especially in major immune cells (B cells, TAMs, NK cells, neutrophils, DCs, MCs, CD8+ T cells and Tregs) (14–16). A considerable part of our research is repeating, verifying, and integrating previously reported results of immune cell phenotypes and functions in PTC. We used ssGSEA, immune cell marker and CIBERSORT to evaluate the infiltration level of immune cells in 10 datasets (a total of 799 PTC and 194 normal samples) from the perspective of abundance and proportion, respectively. Furthermore, it is essential to ensure that the results of the analysis are highly reliable. We also analyzed subtypes of major immune cells and immune cells that lacked attention (such as M1 macrophages, M2 macrophages, Th1, Th2, pDCs, iDCs, eosinophils, monocytes, T_FH cells, T cells gamma delta, and Th17 cells) This will help researchers comprehensively understand the PTC immune microenvironment. Our research supports the evidence that M2 macrophages, Tregs, monocytes, neutrophils, DCs, MCs, and TAMs play a tumor-promoting role, while M1 macrophages, CD8+ T cells, B cells, NK cells, and T_FH cells (eosinophils, γδ T cells, and Th17 cells with weak supporting evidence) exert an antitumor effect. Among them, related reports of the phenotype and function of monocytes, γδ T cells, T_FH cells, and eosinophils in PTC patients are still lacking. Inflammation of monocyte recruitment and activation is associated with the development of TC in a mouse model (74). IL-17-producing γδT17 cells play a decisive role in the chemotherapy-induced anticancer immune response (75). T_FH cells can regulate antibody production by B cells (76); the frequency of T_FH cells increases in patients with autoimmune thyroid disease (AITD), and a significant number of T_FH cells are also detected in the thyroid tissues of patients with Hashimoto’s thyroiditis (77). The above evidence also supports our conclusions about the function of immune cells.

Our research shows that compared with the immune level in normal tissues, the immune level in PTC tissues is generally higher, and the increase in tumor-promoting immune cells is particularly significant. The proportion of immune cells in the TME is also favoring tumor-promoting immune cells. The elevation of TAMs, DCs, MCs, neutrophils, NK cells, and Tregs in PTC has been reported (28–31, 36, 38, 41, 45, 55, 78). Our study found that these tumor-promoting immune cells were recruited in large numbers in PTC and exhibited a tumor-promoting immune microenvironment phenotype. This indicates that PTCs manipulate partial immune cells such as M2 macrophages, Tregs, monocytes, neutrophils, DCs, MCs, and M0 macrophages to fight against the body’s immune response to protect themselves, which is also called “sabotage” (13). Eventually, the tumor evaded immune surveillance and escaped the immune system.

In additional research, we found that immune cell infiltration increased during tumor progression, and the increase in tumor-promoting immune cells was particularly significant. The proportion of immune cells in the TME was further tilted towards tumor-promoting immune cells. Previous mainstream studies have shown that TAMs, DCs, MCs, neutrophils, and Tregs are positively correlated with PTC progression (24, 29–34, 38, 41, 42, 46, 48, 49, 51, 58, 61, 68, 69, 79), and B cells, CD8+ T cells, NK cells, and iDCs are negatively correlated with PTC progression (26, 37, 56, 58–62). Unlike previous studies, our research found that both the abundance and proportion of tumor-promoting immune cells were positively correlated with PTC progression, while the proportion of antitumor immune cells during the progression of PTC decreased (CIBERSORT), and the abundance increased (ssGSEA) or did not significantly change (immune cell marker). These results show that in escalating immune confrontation, immune escape is aggravated, and the tumor’s ability to fight the immune response of the body is further strengthened by recruiting immunosuppressive cells. Eventually, the dynamic balance between antitumor and tumor-promoting immune cells in the original immune system has allowed the irreversible development of tumors. The positive correlations among the H-immunity group, tumor stage, lymph node metastasis, and tumor size also support the above hypothesis.

Our analysis of the correlation of the abundances of immune cells in PTC showed a high positive correlation among all examined cells, indicating that the immune system as a whole has a high degree of consistency. However, antitumor immune cells and tumor-promoting immune cells oppose each other at a proportional level of correlation. Our clustering results on immune cells also show the opposition between immune cells that exert procancer and anticancer effects. We speculate that in the process of tumor progression, the confrontation between tumor-killing immune cells and tumor-controlled immunosuppressive cells in the human immune system continues to escalate, and eventually, the proportion of tumor-promoting immune cells in the TME will have an irreversible advantage. This indicates that the tumor cells escaped the body’s immune surveillance by “sabotage” and achieved immune escape. Except for M0 macrophages (proportion) in our prognosis studies, other immune cells did not reach significant levels. Previous studies in PTC have generally shown that TAMs and neutrophils are associated with a worse prognosis (28, 51–54), while CD8+ T cells and DCs are associated with a better prognosis (26, 39, 62, 63). In fact, the roles of various immune cells in prognosis are still controversial (26, 64), and there are many negative results (36, 48, 50, 64, 70). Considering the positive correlation between immune cell infiltration and tumor progression, we suspect that the insignificant prognosis of immune cells may be related to the lower number of outcomes, such as death, recurrence and metastasis of PTC.

We divided patients into L-immunity and H-immunity groups by consensus clustering and verified the excellent discrimination ability of this grouping for all genes, immune signatures, and TME composition. Compared to patients in the L-immunity group, patients in the H-immunity group showed a more advanced stage, larger tumor size, more lymph node metastasis, higher tall-cell PTC proportions, lower follicular PTC proportions, more BRAF mutations and fewer RAS mutations. The BRAF V600E mutation is related to the immunosuppressive mechanism of PTC; compared to the ratio in wild-type BRAF PTC, the CD8+/FoxP3+ ratio in BRAF V600E mutant PTC was significantly reduced, and the proportion of M2 type TAMs was increased (27, 80, 81). We speculate that somatic mutations in genes such as BRAF and RAS in PTC may initiate the changes in the tumor immune microenvironment.

WGCNA and GSEA were used to explore the underlying mechanisms that cause PTC immune differences, and a number of significantly different immune-related functions and pathways were found. What caught our attention was that Epstein-Barr virus (EBV) infection was the most significantly enriched KEGG pathway. Despite some controversy, many studies have pointed out that EBV is highly expressed in patients with TC and is associated with increasing development of thyroid tumors (82–85). Studies related to nasopharyngeal carcinoma (NPC), Burkitt lymphoma (BL) and gastric cancer have found that EBV can evade host immune recognition and latent infection in B lymphocytes, can epigenetically suppress host tumor suppressor genes, and can provide a potential “hit and run” mechanism for viral carcinogenesis (86–88). We speculate that EBV may play an important role in the change in the PTC immune microenvironment.

However, this study has some limitations. First, ssGSEA, immune cell markers, and CIBERSORT are algorithms based on RNA-seq data, and the abundant results of ssGSEA and immune cell markers are inconsistent in some antitumor immune cells. We have shown their respective results and common conclusions. Second, the research on the mechanism that causes immune differences was based on the inference of several algorithms and has not been experimentally verified.

In the next study, single-cell analysis of immune cells around and within tumors will be an important direction for PTC immune research to elucidate the function of immune cells in the PTC TME.

In conclusion, our research shows that M2 macrophages, Tregs, monocytes, neutrophils, DCs, MCs, and M0 macrophages play a tumor-promoting role in PTC, while M1 macrophages, CD8+ T cells, B cells, NK cells, T_FH cells (eosinophils, γδ T cells, and Th17 cells with weak supporting evidence) play an antitumor role. During the occurrence and development of PTC, the overall immune level increased, and the abundance and proportion of tumor-promoting immune cells significantly increased. We speculate that EBV may play an important role in changing the immune microenvironment of PTC.

The original contributions presented in the study are included in the article/ Supplementary Materials , further inquiries can be directed to the corresponding author/s.

Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

ZX designed the analytical strategies, performed data analyses, and wrote the manuscript. YL, XL, YZH, SW, SYW, JS, and YCH performed data analyses. JZ conceived the research and wrote the manuscript. All authors contributed to the article and approved the submitted version.

This research was supported by the National Natural Science Foundation of China (81600602).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.