Immuno-oncology: The Anti-Tumor Immune Response vs the Anti-Immune Tumor Response

Every time a cell divides, there is potential for introducing mutations that can transform it from healthy to malignant. The immune system is constantly on patrol for these transformed cells, activating an extensive arsenal of anti-tumor immune responses. Innate cells, such as natural killer (NK) cells, NK T cells, and gamma-delta (γδ) T cells are the first line of defence against tumors. Macrophages, dendritic cells (DCs), CD4⁺ T cells, and CD8⁺ T cells act as the last line of defence for eliminating malignant cells.

Meanwhile, tumor cells are engaged in an attack of the immune response. Both intracellular and extracellular metabolic changes create a tumor microenvironment (TME) unfavorable to antitumor immune activity. Tumor development and disease progression results from a failure of the immune response to overcome these attacks.

Navigate to specific cell-type markers using the table below.

Innate Immune Cell Cytotoxicity

NK cells 

NK cells express a combination of activating and inhibitory receptors. The integration of these signals ultimately results in cellular activation and release of cytotoxic molecules.

Bio-Techne has highly validated antibodies for flow cytometry and immunocytochemistry (ICC) applications to detect NK cell markers in both human and mouse cells. 

NKT Cells

NKT cells are activated through recognition of lipid antigens presented to the invariant T cell receptor (iTCR) by CD1d.

γ/δ T Cells

γ/δ T cells express a T cell receptor (TCR) with a gamma chain and delta chain, but do not recognize peptides presented by MHC molecules. Activation of γδ T cells can occur through engagement of the TCR, or by other activation receptors, such as NKG2D. Human γδ T cells expressing a Vγ9Vδ2 TCR recognize phosphoantigens, such as (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) and isopentenyl pyrophosphate (IPP), which are often released by tumor cells. These phosphoantigens bind to CD277/BTN3A1 expressed on the tumor cell and stimulate γδ T cell activation.

Increased prevalence of γδ T cells in mucosal and epithelial tissues, suggest they are strategically positioned for anti-tumor defense. They also present an interesting opportunity for immune cell therapy. One study examining nearly 600 patients with colorectal cancer showed patients with abundant γδ T cells had a higher disease-free 5-year survival rate.

Bio-Techne has a selection of antibodies for general identification of γδ TCR, as well as antibodies to detect specific gamma and delta chains, such as Vγ9 and Vδ2.

The Last Line of Defense: M1 Macrophages, Dendritic Cells, CD4+ and CD8+ T cells

Activation of NK cells, γδ T cells, and NKT cells generates a pro-inflammatory environment which signals to other cells of the immune system that more help is needed. Under these inflammatory conditions, with high levels of IFN-γ, the phenotype of incoming macrophages is skewed toward a tumoricidal, or M1, phenotype. M1 macrophages are associated with a high level of phagocytosis and secretion of pro-inflammatory cytokines that contribute to tumor cell elimination. In addition to canonical macrophage markers, CD11b, CD11c, CD14 and F4/80 (mice), M1 macrophages have distinct expression of surface markers, intracellular factors, and secreted chemokines and cytokines.

Select M1 Macrophage Markers

Dendritic Cells 

Dendritic cells (DCs) are the final bridge between the innate and adaptive immune system. DCs present tumor antigens to naïve CD4+ and CD8+ T cells in the tumor draining lymph nodes and initiate T cell activation. The pro-inflammatory environment created by the NK cells, NKT cells, M1 macrophages, and γδ T cells, results in priming of Th1 helper CD4+ cells and cytotoxic CD8+ T cells.

Immune profiling of tumor infiltrating lymphocytes can provide important information about disease prognosis and potential response to immunotherapy. In addition to a wide selection of highly validated antibodies for flow cytometry, Bio-Techne offers Th1 flow cytometry kits for identification of human and mouse Th1 cells.

Select Markers for CD4+ Th1 and Cytotoxic CD8+ T Cells

Despite the combined antitumor activities of these cells, some tumor cells can escape this elimination process over time, leading to tumor growth and disease progression.

Immunosuppression and Tumor Escape: Going on the Offensive

Tumor cells consistently try to outsmart and outlast the anti-tumor immune response by creating a microenvironment that is hostile to immune cells. The TME consists of multiple different cell types including fibroblasts, endothelial cells, and infiltrating immune cells, whose functions can be exploited or altered to create conditions that are favorable for tumor progression. In addition to hypoxia, the anti-immune tumor response includes differentiation and recruitment of immunosuppressive cells, induction of T cell and NK cell exhaustion, and expression of inhibitory molecules, like PD-L1, HLA-G, and CD47. Many of the molecules implicated in the immunosuppressive mechanisms detailed here are currently being investigated as targets for cancer immunotherapy.

Hypoxia in the TME

Hypoxia is a critical factor to promote immunosuppression in the TME. Commonly measured by HIF-1α expression, tumor hypoxia results from the combined effects of increased cellular proliferation and decreased or sub-optimal blood supply. Episodic or acute hypoxic events are typical in tumors, due to temporary obstruction of blood flow which may limit oxygen supply for short periods of time (minutes to hours). In contrast, as tumors grow, cells are exposed to extended or chronic hypoxia (hours to days), due to abnormal and insufficient vascularization that limits the diffusion of oxygen to some tumor-regions.

Hypoxic tumor cells produce high levels of CCL28, which attracts CXCL10-expressing T_regs. Additionally, HIF-1alpha has been shown to strongly increase the expression of the master T_reg transcription factor forkhead box 3P (FoxP3).

Recruitment and Differentiation of Immunosuppressive Cells in the TME

To create this immunosuppressive environment, tumor cells undergo significant transcriptional and metabolic changes including:

• Increased glucose uptake
• Excess lactate production, leading to low pH in TME
• Increased amino acid uptake
• Increased production of reactive oxygen species (ROS) and reactive nitrogen species (RNS)
• Production of prostaglandin E2 (PGE₂)

These changes in the TME promote differentiation, recruitment, and expansion of immunosuppressive cells such as tumor associated macrophages (TAMs), myeloid derived suppressor cells (MDSCs), and T regulatory cells (T_regs).

Tumor Associated Macrophages (TAMs)

TAMs, sometimes referred to as M2 macrophages, differentiate from monocytes in the presence of immunosuppressive cytokines. Unlike their pro-inflammatory M1 counterparts, M2 macrophages are more likely to express inhibitory ligands like PD-L1, TRAIL, and FASL/TNF6SF and secrete anti-inflammatory cytokines, such as IL-10 and TGF-β. These cytokines are critical mediators of Treg expansion, further M2 polarization, and inhibit NK cell and CD8 T cell activation. TAMs also directly inhibit NK cell cytotoxicity by expressing non-classical MHC I molecules HLA-E and HLA-G.

Selected TAM Markers

Myeloid-Derived Suppressor Cells (MDSCs)

Similar to TAMs, MDSCs are immunosuppressive myeloid cells commonly found in the TME. There are two subsets of MDSCs derived from either a neutrophil (PMN-MDSCs) or a monocyte (M-MDSCs) precursor, with M-MDSCs able to further differentiate into TAMs. In addition to secreting immunosuppressive cytokines, MDSCs pay an active role in altering nutrients and metabolites in the TME. They have elevated expression of metabolic enzymes like iNOS, Arginase 1 (Arg1), and IDO leading to increased reactive oxygen species (ROS), and a decrease in amino acids arginine and tryptophan.

Bio-Techne offers highly validated antibodies to detect and monitor MDSC function for a range of applications including IHC and blocking/neutralization.

Selected MDSC Markers

Regulatory T cells (T_regs)

T_regs are a critical subset of CD4+ T cells that regulate the immune response and prevent autoimmunity. They suppress both Th1 and CD8+ cytotoxic T cells by inducing apoptosis, increasing extracellular adenosine, and inhibiting DC maturation. With increased lactate and hypoxia, the TME is a rich environment for Treg expansion and accumulation. In addition to highly validated antibodies for individual Treg markers, Bio-Techne offers T_reg flow cytometry kits to reproducibly identify T_regs in human, mouse, and rat cells.

Selected T_reg Markers

Cytotoxic T Cell and NK Cell Exhaustion

Cytolytic T cell exhaustion is a hallmark of chronic viral infection and tumor progression. Both CD8+ T cells and NK cells are prone to exhaustion in the TME. CD8+ T cell exhaustion is traditionally understood to be a result of chronic exposure to antigen and sustained signaling through TCR. NK cell exhaustion is also the result of persistent proliferation, decreased signaling from activating receptors and the immunosuppressive environment of the TME. Both are characterized by decreased effector cytokine production, reduced expansion, and upregulation of inhibitory receptors.

Selected Immune Cell Exhaustion Markers

Tumor-derived Extracellular Vesicles

Extracellular vesicles (EVs) are critical mediators of intercellular communication under both physiological and pathological conditions. Small EVs, or exosomes, are implicated in many aspects of cancer progression including tumor growth and metastasis and angiogenesis. Specifically within the TME, small EV-mediated immunosuppressive strategies include inhibiting cytotoxicity of T cells and NK cells, expanding MDSCs and Tregs, as well as inhibition of DC maturation. Due to expression of ligands such as PD-L1, Her-2, and CD20 on the vesicle surface, tumor EVs can serve as decoy targets for immunotherapy, limiting therapeutic efficacy.

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