Within the complex landscape of the tumor microenvironment, populations of immunosuppressive cells constitute a fundamental driver of malignant immune evasion and therapeutic resistance. Through diverse molecular mechanisms, these cellular populations facilitate neoplastic progression, distant dissemination, and circumvention of host immune surveillance, establishing them as critical therapeutic targets in contemporary cancer immunotherapy strategies. The present review examines the classification, mechanistic functions, and regulatory pathways governing the recruitment and activation of three predominant immunosuppressive cell types within the tumor microenvironment.

Major Immunosuppressive Cell Types in TIME

Regulatory T cells

Regulatory T cells (Treg) are a type of T cell subset that controls autoimmune reactivity in the body . They were discovered by Sakaguchi et al. in 1995 and are characterized by the expression of Foxp3, CD25, and CD4. They are T cell subsets other than Th1, Th2, and Th17, and belong to a type of CD4+T cell subset with low proliferation capacity. They have immunosuppressive effects and can secrete cytokines such as IL-4, IL-10, and TGF-B. They play an important role in immune homeostasis and inducing immune tolerance. Treg cells play an important role in infection, tumors, organ transplantation, prevention of autoimmune diseases, and maintaining the body's immune balance.

Origin and classification of Treg cells

According to their surface markers, produced cytokines and action mechanisms, Treg cells can be divided into two categories: one is naturally produced natural Treg cells (nTreg); the other is induced adaptive Treg cells (iTreg). nTreg are mainly CD4+Treg cells, and iTreg include Tr1 cells, Th3 cells and CD8+Treg cells and other subtypes.

Table 1. Types of Tregs.

There are two types of inducing factors for Treg cells: Promotion and Inhibition, which regulate the activation and maturation of Treg cells through induction and inhibition. In tumors, regulatory T cells differentiate from primitive T cells in the thymus through blood circulation. It is now known that regulatory T cells are derived from surrounding primitive T cells induced by tumor microenvironment signals, including tumor antigens, cytokines (such as TGF-β) and some metabolic factors.

Figure 1. The differentiation of Tregs in tumor from naïve T cell in thymus through circulation.

Treg-mediated immunosuppression

Charlotte et al. concluded that Treg cells can inhibit anti-tumor immune responses through multiple mechanisms, mainly including direct cell contact effects and indirect inhibitory pathways mediated by soluble molecules.

Table 2.Treg -mediated immunosuppression.

The role of Treg in the tumor microenvironment

1) Inflammation-related infiltration: chemokine- and cytokine-dependent recruitment

Tregs have multiple chemokine receptors. Chemokine gradients such as CCR4-CCL17/22, CCR8-CCL1, CCR5-CCL5, and CCR10-CCL28 are involved in the recruitment of Tregs into the TME.

2)Metabolic adaptation

Effector T cells and Tregs use different metabolic systems under normal and inflammatory conditions, with Tregs showing a stronger survival advantage.

3)Produce chemokines

EGFR mutations reduce CXCL10 production by inhibiting IFN regulatory factor 1 (IRF1), which negatively affects CXCR3-dependent CD8+ T cell recruitment to the TME.

Myeloid-derived suppressor cells

MDSCs are a heterogeneous cell population, including monocyte-derived MDSCs (M-MDSCs) and granulocyte-derived MDSCs (G-MDSCs). They inhibit the function of T cells and NK cells by producing inhibitory molecules (such as NO and ROS). MDSCs usually exhibit tumor-promoting properties in the tumor microenvironment and may become a potential therapeutic target in immunotherapy .

Origin and classification of MDSCs

MDSCs are diseased neutrophils and monocytes, and MDSCs are mainly divided into two categories: granulocyte-like MDSCs (Granulocytic-MDSCs) and monocytic-like MDSCs (Monocytic-MDSCs). These cells have common surface markers, such as CD11b and CD33, but express different intracellular markers and cytokine patterns, which enable them to play a role in different pathological conditions.

Figure 2. Schematic diagram of myeloid-derived suppressor cell development and differentiation.

MDSC-mediated immunosuppression

MDSCs play a multifaceted immunosuppressive role in tumor immune escape. They systematically weaken anti-tumor immune responses and promote immunosuppressive states in the tumor microenvironment, thereby supporting tumor growth and metastasis, by consuming key amino acids (cysteine, arginine, tryptophan), secreting immunosuppressive factors (such as IL-10, TGF-β), recruiting and activating Treg cells, binding to inhibitory receptors on the surface of T/NK cells, inhibiting NK and DC cell functions, changing macrophage polarization states, and releasing exosomes and chemokines.

Table 3. MDSC- mediated immunosuppression mechanisms

The role of MDSCs in the tumor microenvironment

1)Execution of immunosuppressive functions

Myeloid-derived suppressor cells exert their immunoregulatory effects through multiple complementary mechanisms that collectively compromise anti-tumor immune responses. Through the secretion of potent immunomodulatory mediators, including arginase-1, inducible nitric oxide synthase (iNOS), and reactive oxygen species (ROS), these cells establish direct suppressive effects on both T lymphocyte activation and natural killer cell proliferation. Beyond these direct inhibitory mechanisms, MDSCs orchestrate broader immunosuppressive networks through the facilitation of regulatory T cell (Treg) expansion and the promotion of phenotypic polarization within tumor-associated macrophage (TAM) populations, thereby creating a comprehensive immunosuppressive milieu that supports tumor progression.

2)Recruitment and positioning

MDSCs are recruited to tumor tissues by expressing specific chemokine receptors, such as CCR2 and CCR5, using chemokine gradients such as CCL2 and CXCL12, where they localize and suppress antitumor immune responses.

3)Interaction with tumor cells

MDSCs directly interact with tumor cells and support tumor growth and progression by secreting factors that promote angiogenesis, such as VEGF.

4)Response to treatment

MDSCs play an important role in tumor resistance to chemotherapy and radiotherapy. They can reduce the sensitivity of tumor cells to treatment through multiple mechanisms.

5)Promote tumor immune escape

MDSCs express PD-L1 on their surface, which binds to PD-1 on T cells, further inhibiting the immune system's attack on tumor cells.

Tumor-associated macrophages (TAMs)

Macrophages are a major component of the innate immune system, differentiated from monocytes in the blood, and help the host resist inflammation and tumors. Most tumors can domesticate macrophages into tumor-associated macrophages (TAMs), which promote tumor growth, invasion, and metastasis, and develop resistance to chemotherapy and immune checkpoint inhibitors. However, when properly activated, macrophages can also exert anti-tumor effects by enhancing phagocytosis and cytotoxicity of tumor cells. In addition, TAMs are associated with poor prognosis and resistance to tumor treatment, including immunotherapy, indicating that macrophages are attractive targets for combined therapy in tumor treatment.

Macrophage classification

Based on phenotype and function, macrophages can be divided into two types: classically activated macrophages (M1 macrophages) and alternatively activated macrophages (M2 macrophages).

M1 macrophages can secrete a large number of pro-inflammatory cytokines, such as IL-1β and TNF-α. M1 macrophages are mainly induced by LPS and IFN-γ. Under the action of these substances, macrophages are polarized to M1 type. M1 macrophages participate in positive immune responses by secreting pro-inflammatory cytokines and chemokines and presenting antigens full-time, play the role of immune surveillance, and attack cancer cells. However, excessive pro-inflammatory ability will enhance the inflammatory response and promote the progression of some inflammatory diseases, such as atherosclerosis and severe acute pancreatitis.

M2 macrophages mainly produce anti-inflammatory factors, such as IL-10, TGF-β, and Arg1. M2 macrophages can be activated by IL-4 and IL-13. M2 macrophages have only weak antigen presenting ability and downregulate immune responses by secreting inhibitory cytokines such as IL-10 and/or TGF-B, playing an important role in angiogenesis, promoting tissue repair and wound healing.

Figure 3. Macrophage heterogeneity.

TAMs-mediated immunosuppression

Direct inhibition of T cell function

Direct immunosuppression mediated by TAMs

Tumor-associated macrophages (TAMs) can directly inhibit the anti-tumor function of cytotoxic T lymphocytes (CTLs) through a variety of mechanisms. et al. pointed out that TAMs can highly express immune checkpoint molecules such as programmed death ligand-1 (PD-L1) to inhibit T cell activity. Secondly, they secrete a variety of immunosuppressive cytokines, such as interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), thereby interfering with the proliferation and effector function of T cells. In addition, TAMs further weaken the immune response of T cells by changing metabolic activity, including consuming key metabolic substrates and producing reactive oxygen species (ROS).

Indirect immunosuppression mediated by TAMs

In addition to direct effects, TAMs also indirectly inhibit T cell function by reshaping the tumor immune microenvironment. On the one hand, they can promote the expansion of immunosuppressive cell populations such as regulatory T cells (Tregs), or inhibit the activity of antigen-presenting cells such as dendritic cells (DCs), thereby inhibiting the activation of T cells. On the other hand, TAMs change the tumor vascular structure by regulating the extracellular matrix (ECM) components and chemokine gradients, thereby excluding T cells from the core area of the tumor. In addition, TAMs-induced vascular abnormalities (such as increased expression of ANG2 and VEGFA) will further limit the infiltration and distribution of T cells into tumor tissues, enhancing their immunosuppressive effects.

Techniques to Identify and Quantify Immunosuppressive Cells

The technologies for identifying and quantifying immunosuppressive cells mainly include flow cytometry, mass cytometry (CyTOF), clustering and labeling annotation of single-cell RNA sequencing (scRNA-seq), and CITE-seq combined with protein and transcriptome analysis.

Flow cytometry and CyTOF

Flow cytometry is a widely used technique for phenotyping immune cells by using fluorescently labeled antibodies to detect the expression of specific antigens on the cell surface or within the cell. This technique is capable of detecting multiple antigens simultaneously and performing quantitative and grouped analysis of cells. For example, when identifying myeloid-derived suppressor cells (MDSCs) from human granulocytes and monocytes, flow cytometry can be used to detect specific surface markers, such as CD11b, CD33, etc. In addition, flow cytometry can also be used to detect the secretion of cytokines, such as identifying activated immune cells by detecting specific markers on the cell surface .

scRNA-seq clustering and marker-based annotation

CyTOF is a mass spectrometry-based high-dimensional flow cytometry that uses heavy metal isotope-labeled antibodies to detect protein expression in cells using a time-of-flight mass spectrometer. Compared with traditional flow cytometry, CyTOF avoids the problems of spectral overlap and autofluorescence and can detect up to 100 protein markers simultaneously. CyTOF has significant advantages in immunophenotyping, especially when a large number of protein markers need to be detected simultaneously. For example, in bone marrow samples, CyTOF can simultaneously detect 34 parameters, including cell viability, DNA content, and relative cell size. In addition, CyTOF has also been used to analyze immune signaling pathways, such as changes in cell signaling under drug intervention.

Immune profiling using CITE-seq

CITE-seq (Cellular Indexing of Trans Transcriptomes and Epitopes by Sequencing) is a multimodal technology that combines scRNA-seq and antibody labeling to detect RNA and protein expression simultaneously in the same experiment. CITE-seq detects the expression of cell surface proteins by coupling antibodies to oligonucleotide sequences using PCR amplification and sequencing technology. This method overcomes the limitations of traditional flow cytometry and CyTOF in the number of antibodies and can detect dozens of protein markers simultaneously. An important application of CITE-seq is the identification and characterization of complex immune cell populations. For example, in B cell subsets, CITE-seq can simultaneously detect transcriptome and epitope information, thereby more accurately identifying different B cell subsets. In addition, CITE-seq has also been used to study cellular dynamics in autoimmune diseases, such as identifying resident immune cells in pancreatic islets.

To better understand the distribution and function of immunosuppressive cells in the tumor microenvironment, next-generation sequencing (NGS)-based profiling technologies are increasingly used to complement traditional immunophenotyping. CD Genomics provides NGS-based tumor microenvironment profiling services, enabling high-throughput analysis of immune-related gene expression, cytokine profiles, and cellular compositions.

Conclusion

In summary, understanding the roles of immunosuppressive cells within the tumor immune microenvironment (TME) is essential for advancing cancer immunotherapy. Through this review, I have highlighted the mechanisms by which regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs) contribute to immune evasion, tumor progression, and therapy resistance. These cells suppress antitumor immunity via complex and overlapping pathways involving cytokine secretion, metabolic competition, immune checkpoint engagement, and immune cell recruitment.

Furthermore, I discussed advanced technologies—such as flow cytometry, CyTOF, and CITE-seq—that are instrumental in identifying and characterizing these immunosuppressive populations at high resolution. As research progresses, targeting these cells or their pathways could enhance the efficacy of current immunotherapies and pave the way for more effective combination strategies. A deeper understanding of the immunosuppressive landscape in TME will ultimately bring us closer to overcoming immune resistance in cancer.