Humoral and Cellular Immune Dysregulation and Lung Cancer

Suppression of the Antigen-Presenting Machinery

An adaptive immune response requires two signals between the antigen-presenting cells (APCs) and the effector T cell. The first signal is mediated by the T-cell receptor and the specific antigenic peptide presented in the context of major histocompatibility complex (MHC) class I or class II molecules expressed on the APC surface. The second signal is mediated through constitutively expressed costimulatory molecules on the T cell (CD28) and the APC (B7-1/CD80 or B7-2/CD86; Fig. 16.1 ). The presence of both signals trigger intracellular events resulting in the activation and interleukin-2 (IL-2)-dependent clonal proliferation of T cells.

The MHC class I molecules are essential components of the adaptive immune system and are crucial to the immune recognition of tumor cells. MHC class I molecules report on cellular transformation to CD8 ⁺ cytotoxic T lymphocytes (CTLs) through a multistep process of antigenic peptide acquisition, tagging them for destruction by ubiquitylation, proteolysis, delivery of peptides from cytosol to the endoplasmic reticulum via the heterodimeric transporter associated with antigen processing (TAP) 1 and TAP2 subunits, binding of peptides to MHC class I molecules, and displaying of peptide–MHC class I complexes on the cell surface.

Under physiologic conditions, the components of MHC class I antigen-processing machinery (APM) are constitutively expressed in all adult nucleated cells (except immune privileged tissue). Their expression is regulated by cytokines that can alter the surface expression of MHC class I molecules. Aberrant MHC class I expression has been conclusively demonstrated as an important immune escape mechanism in cancers. MHC class I abnormalities have been frequently found in a variety of human cancers, are associated with unfavorable prognoses in some tumor types, and have a negative impact on the outcome of T cell-based immunotherapy. Marked deficiency or lack of expression of MHC class I molecules has been demonstrated in lung cancer.

The molecular mechanisms of MHC class I expression loss are diverse and include structural alterations or dysregulations of genes encoding the classical MHC class I antigens and/or components of the MHC class I APM. The dysregulation of APM components may occur at the epigenetic, transcriptional, or posttranscriptional level. Mechanisms underlying the aberrant expression of MHC class I antigens in lung cancer include deficiencies in the expression of antigen-processing genes (e.g., genes that encode proteasome subunits and the peptide transporters), which result in defective peptide transport to the cell surface from the endoplasmic reticulum. Haplotype loss of HLA class I antigens is another mechanism of abnormal HLA expression in lung cancer and has been demonstrated in about 40% of lung cancer cell lines. Structural alterations such as β2-microglobulin gene abnormalities resulting from loss of messenger RNA and point mutations are less common mechanisms of altered MHC class I expression in lung cancer.

Studies in which lung cancer cell lines with haplotype loss of HLA class I antigens are used indicate that tumor cells with a normal HLA class I expression may be killed by CTLs at an early stage of carcinogenesis, and only immunoselected tumor cells that lack HLA class I expression can escape this immune attack and develop into cancer. Furthermore, some defects in MHC class I expression in lung cancer, for example, deficiencies in expression of antigen-processing genes and not β2-microglobulin gene abnormalities, may be reversible with cytokines. Interferon-γ ( IFN-γ ) gene transfection into HLA-deficient small cell lung cancer (SCLC) was able to restore its ability to present endogenous tumor antigens to CTL with a concomitant increase in cell-surface expression of class I molecules.

The results of immunohistochemical studies of surgically resected samples with monoclonal antibodies against a common framework determinant of HLA class I antigens have shown deficient expression in HLA class I antigens in 25% to 33% of nonsmall cell lung cancer (NSCLC). As evident from studies of transfected tumor cells lacking expression of these antigens, MHC class I molecules are required for presentation of antigens on tumor cells to the CTL. Thus tumor cells that have lost MHC class I antigens have the advantage of being able to escape lysis by CTL. In lung cancers, the absence of expression of HLA class I molecules has been associated with poor differentiation and aneuploidy, suggesting that lung cancers with abnormal HLA expression may be biologically more aggressive. Taken together, these findings may suggest that tumors with aberrant expression of HLA class I molecules are associated with a poorer prognosis. However, the prognostic implications of HLA class I antigen downregulation in NSCLC are not clear.

Despite expressing antigens recognizable by the host immune system, tumors are very poor at initiating effective immune responses. However, antigen presentation alone is insufficient to activate T cells. In addition to T-cell receptor engagement of an antigenic peptide bound to MHC molecules, additional costimulatory signals are necessary for T-cell activation. The most important of these costimulatory signals is provided by the interaction of CD28 on T cells with its primary ligands B7-1 (CD80) and B7-2 (CD86) on the surface of APCs.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), a member of the immunoglobulin super family and homolog of CD28, binds members of the B7 family with a much higher affinity than CD28. In effect, CTLA-4 competes with CD28 for binding to the B7 family. Upregulation of surface CTLA-4 follows clonal expansion of T cells and regulates the immune response by inducing inhibitory signals in effector T cells that lead to the dampening of the effector T-cell response. CTLA-4 is thus one of the endogenous immune checkpoints that normally terminate immune responses after antigen activation. These T cells are eventually eliminated via apoptosis. CTLA-4 may also contribute to the immune-suppressive function of T-regulatory cells. Upregulation of CTLA-4 on the surface of T-regulatory cells results in suppression of activation and expansion of effector cells specific for both normal self-antigens and tumor antigens. CTLA-4 is constitutively expressed in NSCLC cell lines, where it induces apoptotic cell death on engagement with soluble B7-1 and B7-2 recombinant ligands. Furthermore, CTLA-4 expression in resected early-stage NSCLC was shown to be a good prognostic indicator in a retrospective analysis that was limited by a small number of patients.

Programmed cell death 1 (PD-1) is another key immune checkpoint receptor that is structurally similar to CTLA-4, but with distinct biologic functions and ligand specificity that is expressed by activated T cells and mediates immunosuppression. PD-1 functions primarily in peripheral tissues, where T cells may encounter the immunosuppressive PD-1 ligands PD-L1 (B7-H1) and PD-L2 (B7-DC), which are expressed by tumor cells, stromal cells, or both ( Fig. 16.2 ). The immune inhibitory signals mediated by CTLA-4 and PD-1 are different, as evidenced by the early mortality of CTLA-4 knockout mice compared with modest late-onset strain-specific and organ-specific autoimmunity of PD-1 knockout mice.

Most lung cancer samples, but not samples with normal alveolar cells, express high levels of PD-L1, which is limited to the tumor plasma membrane or cytoplasm. Dendritic cells (DCs) isolated from tumoral and nontumoral lung tissues express low, although significantly higher, levels of B7-1 and B7-2 molecules compared with blood DCs. In surgically resected NSCLC, no relationship was found between the expression of PD-L1 or PD-L2 and clinicopathologic variables, such as histology, stage, or postoperative survival. In a limited subset of patients, fewer tumor-infiltrating lymphocytes (TILs) were identified in PD-L1-positive tumor regions than in PD-L1-negative tumor regions. Tumor-infiltrating and circulating CD8 ⁺ T cells from patients with NSCLC showed increased PD-1 expression compared with peripheral blood mononuclear cells from normal volunteers, but showed impaired immune function, including reduced cytokine production capability and impaired capacity to proliferate. Blocking the PD-1 and PD-L1 pathway by anti-PD-L1 antibodies increased cytokine production and proliferation of PD-1 ⁺ tumor-infiltrating CD8 ⁺ T cells.

Tumor-Derived Soluble Factors

Tumor cells avoid lymphocyte-mediated immune responses by secreting immune inhibitory cytokines in the tumor milieu. In addition to secreting immune-suppressive mediators, tumor cells may also signal surrounding inflammatory cells to release immune-suppressive mediators, augment the trafficking of suppressor cells to the tumor site, and promote the differentiation of effector lymphocytes to a T-regulatory phenotype. Tumor-derived soluble factors also impair T-cell survival. NSCLC cell line supernatants contribute to enhanced activation-induced T-cell apoptosis in the tumor environment. The increased T-cell apoptosis after mitogen stimulation is due to the inhibition of nuclear factor-κB activation in the tumor milieu.