​Mechanism of Infiltration Barrier in Transforming "Cold Tumor" into "Hot Tumor" by Improving T Cell Infiltration (1)

Immunotherapy represented by immune checkpoint inhibitors (ICIs) has greatly improved the clinical efficacy of malignant tumors
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Although ICIs have shown clinical activity in a wide range of tumor types, a substantial proportion of patients do not respond to ICIs therapy
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The antitumor response mediated by ICIs depends on the degree of infiltration of T cells capable of recognizing and killing tumor cells
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Immune cells such as CD8+ T cells have been linked to prolonging the survival of cancer patients and improving the effectiveness of immunotherapy
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Lack of T cells in tumors may lead to resistance to immunotherapy
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The success of CAR-T cell infusion in the treatment of leukemia and lymphoma also demonstrates the importance of T cells in anti-tumor immunity
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According to the spatial distribution of cytotoxic immune cells in the tumor microenvironment (TME), tumors can be classified into one of three basic immune phenotypes: immune inflammatory phenotype, immune rejection phenotype, and immune desert phenotype (Figure 1)
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Immune inflammatory tumors, also known as "hot spot tumors," are characterized by high T cell infiltration, enhanced IFN-γ signaling, and high tumor mutational burden (TMB)
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Tumors with an inflammatory phenotype tend to be more sensitive to ICIs
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Immune rejection tumors and immune desert tumors are called "cold tumors"
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In immunorejected tumors, CD8+ T cells were only located at the edge of invasion and could not effectively infiltrate tumors
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In immune desert tumors, CD8+ T cells are absent in and around the tumor
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In addition to poor T-cell infiltration, "cold tumors" are also characterized by low TMB, low MHC class I expression, and low PD-L1 expression
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Immunosuppressive cell populations, including tumor-associated macrophages (TAMs) and T-regulatory cells (Tregs) and myeloid-derived suppressor cells (MDSCs), are also present in cold tumors
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These features suggest that cold tumors lack innate immune function, or that the innate antitumor immunity present in "cold tumors" may be ineffective due to immune cell rejection
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In contrast to the inflammatory phenotype, cold tumors rarely respond to ICIs monotherapy
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Figure 1.
The three basic immunophenotypes of tumors drive T cell entry into the TME as a gradual process (Figure 2): tumor cell death and antigen release, antigen-presenting cell (APC) processing and presentation of tumor antigens, APC and T Cellular interactions lead to T cell priming and activation
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Ideally, once activated, these tumor-specific T cells would leave the lymph nodes and travel through the bloodstream to the tumor site
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The generation of T cells and their physical contact with tumor cells is the key to the success of antitumor immunity
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Once inside the tumor bed, CTLs can specifically recognize the antigenic peptide-MHC complexes on the surface of tumor cells, form immune synapses, and release perforin and granzymes or Fas/FasL pathway to destroy tumor cells
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Dead tumor cells release additional tumor antigens, thereby amplifying the T cell response
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Figure 2.
Processes that drive T cells into the TME Given the importance of T cell infiltration, it is necessary to understand the mechanisms by which T cells homing to tumors
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To enhance the clinical benefit of immunotherapy, ICIs can be combined with strategies to convert "cold tumors" into "hot tumors", which can make these tumors more responsive to ICIs therapy
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This article will provide a brief summary of the various mechanisms of T-cell infiltration disorders and strategies for T-cell infiltration of tumors in two articles
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ICIs response rates are low in "cold tumors," characterized by the absence of T-cell infiltration
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In driving T cells into tumors, many factors can influence T cell priming and T cell homing to the tumor site, resulting in a non-inflammatory phenotype of T cells and failure of antitumor immunity (Figure 3)
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Figure 3.
Mechanisms of three different tumor phenotypes.
Defective T cell priming Table 1 summarizes the main mechanisms of defective T cell priming
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Table 1.
Major Mechanisms of Deficient T Cell Priming Lack of Tumor Antigens Due to the lack of tumor antigens, the most immediate cause of impaired T cell priming is insufficient T cell recognition
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In general, targeting tumor antigens can be divided into two broad categories: non-mutated self-antigens and neoantigens resulting from non-synonymous somatic mutations
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Autoantigens include non-mutated proteins that are aberrantly or overexpressed in tumor cells, such as tumor-associated antigens (TAAs) and cancer/testis antigens (CTAs)
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Although autoantigens also elicit tumor immune responses, the primary targets of immune responses are neoantigens, also known as tumor-specific antigens (TSAs)
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Neoantigens arise from somatic mutations in the cancer genome and are therefore specific to tumor cells
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Recognition of tumor neoantigens may promote T cell initiation and infiltration and lead to long-term clinical responses
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TMB is broadly described as the total number of non-synonymous single nucleotide mutations in a tumor
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In general, tumors with higher levels of TMB are thought to carry higher neoantigen loads that can be recognized by T cells, making them more likely to prime the immune system
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In various tumor types, a significant association between high TMB and improved response to ICIs has been reported
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TMB has been used as a novel biomarker to predict the efficacy of PD-1/PD-L1 inhibitors
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Consistent with the importance of the efficacy of ICIs, high TMB was associated with greater immune cell infiltration
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Defects in tumor antigen processing and presentation machinery (APM) After recognizing tumor antigens, APCs process the antigen and express the corresponding antigenic peptide-MHC class I complexes on their surface
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However, alterations in APM, such as downregulation of MHC-I molecule expression or deletion of β-2-microglobulin (B2M), limit the presentation of antigenic peptide-MHC class I complexes
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In addition, the lysosomal pathway was associated with reduced CD8+ T lymphocyte infiltration
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In PDAC, the autophagy-related receptor NBR1 induces the degradation of MHC-I on the tumor cell surface, which in turn affects T cell responses
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These findings suggest that defects in tumor antigen processing and presentation pathways inhibit T cell priming and the effectiveness of cancer immunotherapy
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Dysfunction of DC-T cell interaction Dendritic cells (DCs) are a specialized class of APC cells with unique abilities to acquire antigens, migrate to secondary lymphoid organs such as lymph nodes and spleen, and initiate immune responses in vivo
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DC activation requires pattern recognition receptors (PRRs) on their surface to recognize "danger signals", including pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs)
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This recognition enables DCs to present tumor antigen peptide-MHC-class I complexes to T cells upon contact with T cells
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DCs also express co-stimulatory signals, such as B7 (including CD80 and CD86), which provide secondary signals required for T cell activation
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Tumor cells can mediate attenuated DC phagocytosis by capturing "danger signals"
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For example, the intracellular checkpoint stanniocalcin 1 (STC1) can trap blockers such as calreticulin (CRT), thereby inhibiting DC phagocytosis and T cell activation, contributing to tumor immune escape
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In melanoma patients, STC1 is associated with low T cell activity and survival
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DC cells are generally divided into two categories: plasmacytoid dendritic cells (pDCs) characterized by the production of IFN-α and conventional dendritic cells (cDCs) that can effectively stimulate the proliferation of T cells
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CDCs are further divided into two distinct subsets: BATF3-dependent DCs and IRF4-dependent DCs
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BATF3 dendritic cells are able to cross-present tumor-derived antigens through the MHC-I pathway, thereby priming T cells
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In addition, BATF3 dendritic cells are a major source of CXC-chemokine ligand 9 (CXCL9) and CXCL10, key chemokine ligands that recruit CXCR3-expressing CD8+ T cells to tumors
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In melanoma, the expression of BATF3 DC markers (eg, BATF3 and IRF8), CXCL9, CXCL10, and CXCL11 was significantly correlated with the CD8+ effector T cell phenotype
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In the absence of BATF3 dendritic cells, CD8+ effector T cells are unable to migrate into tumors and thus have defective antitumor immunity
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This finding confirms that BATF3 dendritic cells may be required for the initiation and recruitment of endogenous T cells to fight tumors
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Regulation of Fms-like tyrosine kinase 3 ligand (FLT3L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) is important for DC differentiation and recruitment
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FLT3L is a growth factor that promotes the differentiation of hematopoietic progenitor cells from the bone marrow to the DC lineage
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Tumor-derived FLT3L increased infiltration of BATF3 dendritic cells and CD8+ T lymphocytes in mouse tumors and increased migratory and resident DC subsets in draining lymph nodes, suggesting that FLT3L has a mobilizing effect on DC cells
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Lack of FLT3L or GM-CSF results in reduced numbers of DCs in secondary lymphoid organs and attenuated T-cell immune responses
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Given the important role of DC-T cell crosstalk in T cell priming, impaired DC activation, DC deficiency, and overexpression of co-inhibitory signals can lead to impaired T cell activation
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Defects in T cell homing to the tumor Table 2 summarizes the mechanisms that prevent T cell homing to the tumor bed
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Table 2.
Mechanisms that prevent T cells from homing to tumor beds.
Oncogenic pathway activation There is growing evidence that activation of oncogenic pathways in tumor cells is associated with the "cold tumor" phenotype and the likelihood of immunotherapy resistance
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In Wnt/β-catenin-positive melanoma, decreased CCL4 production leads to decreased recruitment of BATF3 DCs to the TME
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Ultimately, in the absence of CXCL9 and CXCL10 produced by BATF3 DCs, CTLs were not recruited to tumors
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An analysis of human metastatic melanoma samples revealed that CD8A expression was inversely correlated with activation of the β-catenin signaling pathway
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Direct injection of BATF3 DCs helped restore T-cell infiltration in β-catenin-positive tumors and resulted in tumor suppression
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This result suggests that activation of Wnt/β-catenin signaling and deficient BATF3 DCs mediate T cell rejection and tumor cell escape from the immune system
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Loss of PTEN activates the PI3K/AKT pathway, which is associated with a non-inflammatory T cell phenotype and immune tolerance in melanoma
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Loss of PTEN expression has been found to reduce lipidation of the autophagy protein LC3, resulting in decreased autophagic activity, thereby inhibiting T cell initiation and T cell-mediated antitumor responses
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CD8+ T cell infiltration was significantly reduced in PTEN-deficient melanomas compared with PTEN-expressing melanomas
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The results of the TCGA dataset analysis showed that the expression of T-cell effector molecules such as IFN-γ and granzyme B was significantly reduced in melanomas with low PTEN expression
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As the most commonly mutated gene associated with cancer progression, RAS leads to the activation of multiple signaling pathways, such as MAPK and PI3K, that drive tumorigenesis
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Additionally, oncogenic K-RAS mutations mediate inflammation and crosstalk with the TME
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For example, oncogenic K-RAS mutations induce pro-tumor inflammation through production of inhibitory cytokines (eg, IL-6 and IL-8), activation of the NLRP3 inflammasome, and release of chemokines (eg, CCL5 and CCL9)
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Oncogenic signaling through MYC enhances the expression of CD47 and PD-L1 on tumor cells
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CD47 binds to the inhibitory receptor Sirpα on the surface of APC cells such as macrophages and dendritic cells, which can prevent the phagocytosis of tumor cells and interfere with antigen uptake
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Oncogenic KRAS and MYC synergistically induce immune modulation
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For example, in a mouse model of lung cancer, co-activation of KRAS and MYC resulted in the production of CCL9 and IL-23
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It mediates interstitial reprogramming, promotes angiogenesis, and excludes T, B, and NK cells from tumors
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Activation of CDK4/6 and STAT3 was also associated with a non-inflammatory T cell phenotype
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Taken together, these results suggest that activation of oncogenic pathways can affect not only tumor cells but also T cell-mediated antitumor immunity
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Chemokines and their epigenetic regulation Interactions between certain chemokine receptors on effector T lymphocytes and the corresponding chemokines may influence the transport of effector T lymphocytes to tumor sites
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Deficiencies of several chemokines, including CXCL9, CXCL10, CCL4, CCL5, CXCL16, CX3CL1, have been reported to lead to T cell rejection
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Given the importance of the TH1-type chemokines CXCL9 and CXCL10 for T cell recruitment, some tumors showed low levels of CXCL9 and CXCL10 expression, possibly explaining the decreased infiltration of effector T lymphocytes in these tumor beds
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For example, BATF3 dendritic cells are the main source of CXCL9 and CXCL10, and lack of BATF3 dendritic cells results in low expression of CXCL9 and CXCL10
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In addition, epigenetic regulation of tumors is also important for maintaining low expression levels of these cytokines
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DNA methyltransferase (DNMT) and ZEST homologous enhancer 2 (EZH2) mediate DNA methylation and histone lysine methylation, respectively, thereby inhibiting the expression of CXCL9 and CXCL10 in ovarian cancer
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Similar results were also confirmed in colon cancer
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In addition to CXCL9 and CXCL10, CCL5 expression was positively correlated with CD8+ T cell infiltration
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Binding of CCL5 to CCR5 promotes the recruitment of CD8+ T cells
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However, DNA methylation resulted in loss of CCL5 expression, which in turn resulted in loss of CD8+ T cell infiltration
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In a mouse model of NSCLC, the combination of a DNMT inhibitor and a histone deacetylase (HDAC) inhibitor increases the expression of endogenous retrovirus (ERV), which induces a type I interferon response
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This combination therapy reversed immune tolerance in a NSCLC model by downregulating oncogenic MYC signaling, resulting in increased CCL5 and enhanced T cell infiltration into tumors
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Some chemokines are detrimental to T cell trafficking to tumors
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Stromal cells, especially cancer-associated fibroblasts (CAFs), are the main producing cells of CXCL12
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CXCL12 produced by CAFs misdirects CTL to the extratumoral stroma and prevents CTL from entering the tumor
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Furthermore, high expression of CXCL8 has been reported to be associated with reduced T cell numbers, increased neutrophil and monocyte infiltration, and limited response to ICIs in tumors
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These results reveal the regulatory role of chemokine receptor and ligand interactions on CTL homing to tumors and their integration into the TME
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Abnormal vasculature and adequate infiltration of hypoxic T cells in tumors not only depend on the recruitment of appropriate chemokines, but are also controlled by tumor vasculature
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During the process of CD8+ T lymphocyte metastasis to the tumor, they must enter the tumor circulatory system, adhere to the vascular endothelial cells, and migrate through the vascular wall
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Recruitment of CD8+ T cells in tumors requires the action of vascular endothelial cell adhesion molecules, including P- and E-selectins, intercellular adhesion molecules (ICAMs), and vascular cell adhesion molecules (VCAMs)
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However, downregulation or ineffective aggregation of adhesion molecules on tumor endothelial cells results in endothelial anergy and reduced T cell trafficking to the tumor site
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Endothelin binds to the endothelin B receptor (ETBR) on endothelial cells and reduces the production of ICAM-1, thereby inhibiting the adhesion of CD8+ T cells to endothelial cells
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In addition, vascular endothelial growth factor (VEGF) produced by tumor and mesenchymal cells stimulates endothelial cell proliferation, leading to the formation of new blood vessels, often accompanied by impaired tissue perfusion and increased vascular permeability
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VEGF can also reduce the expression of important molecules such as VCAM-1 on the endothelial cell surface, ultimately preventing T cells from migrating to the TME
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Another mechanism by which tumor endothelial cells inhibit T cell migration is the modulation of immune cell activity
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IL-10, PGE2 and VEGF can induce the up-regulation of FasL expression in tumor endothelial cells, thereby killing tumor-associated T cells, and anti-FasL can attenuate this killing effect
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Acetylsalicylic acid (ASA) and VEGF antibodies that inhibit COX and PGE2 activity promote the infiltration of CD8+ T lymphocytes in the TME and improve prognosis
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In addition, impaired vascular tight junctions and increased permeability lead to hypoxia, acidosis, and necrosis, which inhibit immune effector T cell function and antitumor immunity
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A hallmark of cancer, hypoxia is caused by increased oxygen demand due to tumor cell proliferation and insufficient blood supply due to angiogenesis
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Hypoxia-inducible factor 1 (HIF1) is a key transcription factor activated by hypoxia
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Hypoxia inhibits T cell infiltration in several ways
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First, hypoxia promotes the recruitment of immunosuppressive cells to the TME
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Second, hypoxia-induced CCL28 and VEGF promote angiogenesis and affect T cell trafficking
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Finally, hypoxia and TGFβ can upregulate the expression of CD39 and CD73 in tumor tissue
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CD39 and CD73 catalyze the sequential conversion of ATP to extracellular adenosine (ADO)
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Adenosine binds to the adenosine A2A receptor (A2AR) and inhibits the production of cytokines such as IL-2, thereby inhibiting the development and proliferation of T cells
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In a mouse melanoma model, A2AR inhibition increased T lymphocyte infiltration and resulted in improved tumor control, suggesting a potential role of ADO signaling in promoting T cell rejection
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In addition, ADO can also promote the recruitment and polarization of MDSCs and Treg cells by inhibiting the effector functions of NK cells and DCs, thereby impairing antitumor immunity
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TME: Immunosuppressive Cells and Factors The immunosuppressive microenvironment at tumor sites, including dense stroma and immunosuppressive cells and factors, can prevent T cell priming and infiltration in "cold tumors"
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TGFβ is a potent immunosuppressive cytokine that promotes immune escape and prevents the acquisition of a TH1 effector phenotype
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The major producer of TGFβ is the abundant CAF in the TME
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Increased TGFβ production by CAF is associated with T cell rejection in tumors and poor response to PD1/PDL1 mAbs
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TGFβ restricts the proliferation of CD4+ T lymphocytes by inhibiting the production of IL-2 and induces the transformation of naive CD4+ T lymphocytes into Tregs
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TGFβ also negatively affects DC differentiation and antigen-presenting functions, which interfere with T cell priming
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In conclusion, TGFβ hinders anti-tumor immunity by affecting T cell differentiation and function, preventing T cells from infiltrating tumors
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Tryptophan metabolism is frequently dysregulated in a variety of cancers and has been implicated in immune tolerance
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Indoleamine 2,3-dioxygenase (IDO) in tumor cells can convert the essential amino acid tryptophan to kynurenine, thereby blocking the initiation of T lymphocytes and promoting the development of Tregs
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IDO also recruits and activates MDSCs and inhibits tumor-specific T lymphocyte accumulation in tumors
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However, the failure of the IDO inhibitor epacadostat in combination with Keytruda in the Phase III ECHO-301 study suggests that the efficacy of IDO-targeting drugs requires further consideration
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CAFs are key cellular components in the tumor stroma that promote tumor growth
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Mainly located at the infiltrating margin of tumors, CAFs regulate tumor metastasis and influence angiogenesis by synthesizing and remodeling the extracellular matrix (ECM) and producing cytokines, and transforming the tumor margin into an immune "cold" zone
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CAFs lead to immunosuppression and T cell rejection through several mechanisms
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First, CAFs create an extracellular matrix that forms a physical barrier that prevents T cells from infiltrating the tumor area
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Second, CXCL12 produced by CAFs has been shown to inhibit T lymphocyte infiltration within tumors in pancreatic cancer models
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Third, CAFs can also reduce T cell responses and exert immunosuppressive effects by producing TGFβ and IL-6
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Reprogramming the CAF is an effective strategy to achieve "normalization" of the TME
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This strategy reduces extracellular matrix levels, reduces vascular stress, and increases T cell penetration, thereby improving cancer therapy
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Furthermore, TAMs keep T cells out of tumors by modulating the ECM and mediating CCL2 and CCL5 signaling
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TAMs promote abnormal angiogenesis by producing VEGF and matrix metalloproteinase-9 (MMP9), thereby affecting the recruitment of T cells
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The interaction of cytokine colony-stimulating factor-1 (CSF-1) and CSF-1R promotes the differentiation of myeloid cells into an immunosuppressed M2 macrophage phenotype
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Targeting TAMs with CSF1R inhibitors reduces the number of TAMs and increases the infiltration of effector lymphocytes such as CD8+ T cells
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Tumor cells are typically characterized by high rates of glucose uptake and active glycolysis, even in the presence of oxygen
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This phenomenon is called the "Warburg effect"
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During this process, glucose is rapidly consumed and the abundance of lactate in the TME increases
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The glucose-deficient, lactate-rich TME exerts metabolic stress on infiltrating T cells, leading to local immunosuppression and ICI tolerance
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Glucose-deficient metabolism in the TME mediates T cell hyporesponsiveness, inhibits mTOR activation, and reduces glycolytic capacity and INFγ production
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Furthermore, glycolytic activity and T cell infiltration were inversely correlated in a variety of tumors
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Consistent with this, high expression of glucose transporter 1 (GLUT-1) in renal cell carcinoma was associated with low infiltration of CD8+ T cells
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These results suggest that glycolytic tumors are associated with a non-inflammatory T cell phenotype
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In addition to tumor cells, stromal cells, such as CAFs and TAMs, can also promote the accumulation of lactate in the TME
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Lactic acid accumulation and acidification of the TME suppresses antitumor immunity
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Lactic acid-induced acidosis impairs monocyte-to-dendritic cell differentiation and inhibits the antigen-presenting function of dendritic cells, which in turn inhibits T cell activation
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Furthermore, lactate inhibits the chemotactic and antitumor activities of CTLs and promotes tumor immune escape
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Inhibiting lactate production or restoring the physiological pH of the TME reversed the inhibitory effect of lactate on antitumor immunity
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For example, neutralization of tumor acidity with sodium bicarbonate, combined with ICIs or adoptive cell therapy (ACT), can effectively promote T cell infiltration and improve antitumor responses in various mouse tumor models
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In addition to glucose, metabolic competition between tumor and immune cells includes amino acids and fatty acids
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For example, rapid esterification of cholesterol in tumors inhibits T cell receptor (TCR) aggregation and immune synapse formation
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ACAT1 inhibitor avasimibe, the key enzyme of cholesterol esterification, can promote the proliferation of CD8+ T cells and has a good anti-tumor effect
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The new study also confirmed that inhibition of PCSK9, a key protein that regulates cholesterol metabolism, can upregulate MHC-I levels on the surface of tumor cells, increase CTL infiltration within tumors, and synergize with anti-PD1 antibodies to inhibit tumor growth
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Considering the interplay between tumor metabolism and immune cell metabolism, directing metabolic pathways to reduce metabolic stress on T cells is a promising strategy to improve the efficacy of immunotherapy
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References 1.
Turning cold tumors into hot tumors by improving T-cell infiltration (main literature).
2.
Primary, adaptive, and acquired resistance to cancer immunotherapy.
3.
Approaches to treat immune hot, altered and cold tumors with combination immunotherapies.