Numerous immune cells coexist in the TME, including T and B lymphocytes, macrophages, polymorphonuclear cells, mast cells, natural killer cells, dendritic cells and myeloid-derived suppressor cells (MDSCs). These cell clusters can infiltrate into the TME and alter it after targeted therapy (6).

During the response period to TKIs, CD8⁺ T cells and dendritic cells expand, while immunosuppressive cells such as Foxp3⁺ regulatory T cells (Tregs) and M2-like polarization of macrophages are inhibited. T-cell infiltration in the TME is elevated by TKIs (8). Increased lymphocyte infiltration generates a local inflammatory TME. For example, skin rash is a common side effect of EGFR-TKIs. EGFR-TKIs have been reported to alter the levels of circulating cytokines and lymphocytes (9). A significant increase in peripheral natural killer cells and interferon-γ (IFN-γ) levels is observed after 4 weeks of gefitinib treatment. In addition, erlotinib and gefitinib improve the susceptibility of cancer cells to natural killer cells (10). EGFR inhibitors induce the expression of major histocombatibility complex (MHC) class I and II molecules and promote T cell-mediated tumor death (11). Circulating interleukin 6 (IL-6) levels are significantly decreased, which might predict better progression-free survival (7). EGFR TKIs decrease pro-inflammatory cytokines such as CC chemokine ligand (CCL)2, CCL5, C-X-C motif chemokine ligand (CXCL)8, CXCL10, and IFN-γ-induced protein 10, and increase the level of IFN-γ (9,12).

In mouse models with sensitive EGFR mutations, erlotinib increases the infiltration of T lymphocytes and natural killer cells other than B cells. Dendritic cells and macrophages with increased MHC class II expression may enhance antigen-presenting activity. The levels of immune checkpoints in the TME were reduced (13). Several EGFR downstream signaling pathways [IL-6/Janus kinase (JAK)/STAT3, NF-κB, and phospho-ERK1/2/phospho-c-Jun] regulate PD-L1 expression in tumor cells (3,7). An NSCLC specimen study illustrated that PD-L1 expression was downregulated after 4 weeks of gefitinib treatment (14). EGFR TKI treatment led to a decrease in PD-1, cytotoxic T lymphocyte-associated antigen 4 and T cell immunoglobulin and mucin domain-containing protein 3 expression in T cells in mouse models (8). EGFR TKIs and anti-PD-1 antibody combination treatment produced no synergistic antitumor effect in an in vitro co-culture system (3). Reduced expression of immune checkpoints within the TME may explain these clinical observations (15).

ALK inhibitors have been reported to increase T-lymphocyte infiltration. Recently, whole-exome and RNA sequencing indicated that immune-related genes were altered in response to ALK TKI treatment. Antigen presentation genes, IFN-γ signaling genes, inhibitory checkpoint genes, and stimulatory checkpoint genes increased apparently after response to ALK TKIs (Table I) (16).

In conclusion, short-term exposure to TKIs not only eliminates tumor cells, but also modulates immune-mediated cytotoxicity in the TME during the initial response period. One study provided a rationale for the potential combination of erlotinib and immunotherapies for the treatment of lung carcinomas in first-line treatment (17). However, other pre-clinical studies confirmed that synergistic effects of EGFR-TKIs combined with anti-PD-1 antibody were not present in an in vitro co-culture system (3). According to the latest edition of the National Comprehensive Cancer Network guidelines for NSCLC (18), PD-1/PD-L1 inhibitor may not be recommended for EGFR⁺/ALK⁺ NSCLC due to the negative results generated in a retrospective study (19). Therefore, TKI monotherapy is preferred for oncogene-driven NSCLC in the first-line treatment.

A decrease in cytotoxic T cell populations was observed in EGFR inhibitor-resistant lung adenocarcinoma when compared with that in the initial biopsy from an identical patient (22). Some resistant tumors are deficient for MHC class I as a result of decreased mRNA levels and related genes (23). Thus, targeted treatment may influence different aspects of tumor antigen presentation and T-cell effector function. Mitogen-activated protein kinase is the most common compensatory signaling pathway responsible for the development of acquired resistance (24). In the examination of tumor biopsies, cytotoxic T cells are found to decrease after TKI resistance and T-cell infiltration. Tregs are the main component of the TME. Conversely, the number of macrophages, especially those expressing indoleamine 2,3-dioxygenase 1 (IDO1), increase with disease progression (25).

MDSCs are immature myeloid cells that mainly inhibit T-cell activation; they help create a favorable environment for tumor survival. In an in vivo study an increased percentage of MDSCs was detected during long-term use of EGFR-TKI in mouse models (8,26). Recruitment and expansion of MDSCs were detected in the serum of patients with NSCLC who achieved EGFR TKI resistance, compared with the baseline (27). In the TME, MDSCs impair host antitumor immunity and the effect of immunotherapy (28).

PD-L1 expression levels in tumor cells were found to notably increase after gefitinib treatment in a subset of patients (29). PD-L1 is a well-known immune checkpoint gene, and knockout of PD-L1 helps to restore the function of human cytotoxic T lymphocytes (30,31). The histological transformation of the tumor and metastasis sites affects the PD-L1 expression levels after resistance (32). In one study, paired analysis indicated that tumors with TPS ≥50% increased from 14 to 28% after acquired resistance, especially obvious in the EGFR T790M-negative tumor (31). High expression levels of PD-L1 have been reported to be a negative prognostic marker (33) and although lung cancer cells develop resistance to EGFR TKIs, EGFR phosphorylation is still suppressed in the resistant cells. However, the downstream ERK signaling is reactivated upon drug resistance, leading to PD-L1 restoration (34,35). Several studies have revealed that PD-L1 is a downstream target of the EGFR pathway, which is interceded via the IL-6/JAK/STAT3, NF-κB, and phospho-ERK1/2/phospho-c-Jun pathways (3,36–38). Single-cell RNA sequencing of biopsy samples showed that immunosuppressive genes, such as IDO1, kynureninase and quinolinate phosphoribosyltransferase levels were elevated in TKI-resistant tumors compared with those in naïve tumors (25). Dynamic changes are shown in Figure 1.

Limited data have been reported on the impact of ALK inhibitors on the TME after resistance in ALK-positive NSCLC. A preclinical study explored the changes in the tumor immune microenvironment in mouse models. The results indicated that ALK-positive NSCLC was as an immune ‘desert’ before the initiation of targeted therapy (39). After resistance to ceritinib the immunogenic features of TME changed, including increased PD-L1 expression levels, increased number of functionally impaired CD8⁺ T cells, antigen-presenting cells, Tregs and MDSCs, and increased IFN-γ secretion. RNA sequencing analysis revealed increased transcription of IFN-γ-related genes, Treg-related genes and immune suppressive macrophage-related genes. However, whole-exome sequencing revealed no obvious difference in tumor mutation burden and T cell receptor clonality between untreated and resistant ALK-positive tumors (39). Thus, due to the lack of immunogenicity and impaired antitumor immunity, ICIs are expected to be less effective in ALK-TKI-resistant tumors.

In conclusion, along with the emergence of acquired resistance, the TME begins to gain immunosuppressive features, such as the presence of inhibitory ligands, restoration of the MDSCs and Tregs, impaired functions of tumor-infiltrating lymphocytes, decreased antigen presentation, increased level of IFN-γ and increased immune checkpoint molecules (Table I) (8). Subsequently, immune evasion and T-cell exhaustion lead to disease progression (40). The potential mechanisms that are involved in these changes include: i) The reactivation of EGFR downstream signaling pathways; ii) the secretion of tumor-related exosomes by EGFR-mutated cells; and iii) the induction of MHC expression. The change in the TME indicates the possibility of initiating combination strategies in resistant patients.