Chien-Chung Chang

Immune selective pressure and HLA class I antigen defects in malignant lesions

The revived cancer immune surveillance theory has emphasized the active role the immune system plays in eliminating tumor cells and in facilitating the emergence of their immunoresistant variants. MHC class I molecule abnormalities represent, at least in part, the molecular phenotype of these escape variants, given the crucial role of MHC class I molecules in eliciting tumor antigen-specific T cell responses, the high frequency of HLA class I antigen abnormalities in malignant lesions and their association with a poor clinical course of the disease. Here, we present evidence for this possibility and review the potential mechanisms by which T cell selective pressure participates in the generation of tumor cells with MHC class I molecule defects. Furthermore, we discuss the strategies to counteract tumor cell immune evasion.

HLA class I antigen loss, tumor immune escape and immune selection.

Poor clinical response rates have been observed in the majority of the T cell-based immunotherapy clinical trials conducted to date. One reason might be the presence of abnormalities in HLA class I antigen presentation in malignant lesions. An increased frequency of HLA class I abnormalities has been observed in malignant lesions from patients treated with T cell-based immunotherapy and in lesions which have recurred in patients who had experienced clinical responses following T cell-based immunotherapy. These observations are compatible with the possibility that the outgrowth of a patients tumor reflects immune selection of tumor cells which have acquired escape mechanisms from immune recognition.

Sequential Immune Escape and Shifting of T Cell Responses in a Long-Term Survivor of Melanoma

Immune-mediated control of tumors may occur, in part, through lysis of malignant cells by CD8+ T cells that recognize specific Ag-HLA class I complexes. However, tumor cell populations may escape T cell responses by immune editing, by preventing formation of those Ag-HLA complexes. It remains unclear whether the human immune system can respond to immune editing and recognize newly arising escape variants. We report an example of shifting immune responses to escape variants in a patient with sequential metastases of melanoma and long-term survival after surgery alone. Tumor cells in the first metastasis escaped immune recognition via selective loss of an HLA haplotype (HLA-A11, -B44, and -Cw17), but maintained expression of HLA-A2. In the second metastasis, immune escape from an immunodominant MART-1-specific T cell response was mediated by HLA class I down-regulation, resulting in a failure to present this epitope, but persistent presentation of a tyrosinase-derived epitope. Consequent to this modification in tumor Ag presentation, the dominant CTL response shifted principally toward a tyrosinase-targeted response, even though tyrosinase-specific CTL had been undetectable during the initial metastatic event. Thus, in response to immune editing of tumor cells, a patient’s spontaneous T cell response adapted, gaining the ability to recognize and to lyse “edited” tumor targets. The observation of both immune editing and immune adaptation in a patient with long-term survival after surgery alone demonstrates an example of immune system reactivity to counteract the escape mechanism(s) developed by tumor cells, which may contribute to the clinical outcome of malignant disease.

Classical and Nonclassical HLA Class I Antigen and NK Cell–Activating Ligand Changes in Malignant Cells: Current Challenges and Future Directions

Changes in classical and nonclassical HLA class I antigen and NK cell-activating ligand expression have been identified in malignant lesions. These changes, which are described in this chapter, are believed to play a major role in the clinical course of the disease since both HLA class I antigens and NK cell-activating ligands are critical to the interaction between tumor cells and components of both innate and adaptive immune systems. Nevertheless, there is still debate in the literature about the biologic and functional significance of HLA class I antigen and NK cell-activating ligand abnormalities in malignant lesions. The reasons for this debate are reviewed. They include (i) the incomplete association between classical HLA class I antigen changes and the clinical course of the disease; (ii) the relatively limited number of malignant lesions that have been analyzed for nonclassical HLA class I antigen and NK cell-activating ligand expression; and (iii) the conflicting data regarding the role of immunoselection in the generation of malignant cells with HLA antigen and NK cell-activating ligand abnormalities. The technical limitations associated with the assessment of HLA antigen and NK cell-activating ligand expression in malignant lesions as well as the immunological and nonimmunological variables that may confound the impact of HLA antigen and NK cell-activating ligand changes on the clinical course of the disease are also discussed. Future studies aimed at overcoming these limitations and characterizing these variables are expected to provide a solution to the current debate regarding the significance of HLA class I antigen and NK cell-activating ligand abnormalities in malignant lesions.

Immune Selection of Hot-Spot β2-Microglobulin Gene Mutations, HLA-A2 Allospecificity Loss, and Antigen-Processing Machinery Component Down-Regulation in Melanoma Cells Derived from Recurrent Metastases following Immunotherapy

Scanty information is available about the mechanisms underlying HLA class I Ag abnormalities in malignant cells exposed to strong T cell-mediated selective pressure. In this study, we have characterized the molecular defects underlying HLA class I Ag loss in five melanoma cell lines derived from recurrent metastases following initial clinical responses to T cell-based immunotherapy. Point mutations in the translation initiation codon (ATG→ATA) and in codon 31 (TCA→TGA) of the β2-microglobulin (β2m) gene were identified in the melanoma cell lines 1074MEL and 1174MEL, respectively. A hot-spot CT dinucleotide deletion within codon 13–15 was found in the melanoma cell lines 1106MEL, 1180MEL, and 1259MEL. Reconstitution of β2m expression restored HLA class I Ag expression in the five melanoma cell lines; however, the HLA-A and HLA-B,-C gene products were differentially expressed by 1074MEL, 1106MEL, and 1259MEL cells. In addition, in 1259MEL cells, the Ag-processing machinery components calnexin, calreticulin, and low m.w. polypeptide 10 are down-regulated, and HLA-A2 Ags are selectively lost because of a single cytosine deletion in the HLA-A2 gene exon 4. Our results in conjunction with those in the literature suggest the emergence of a preferential β2m gene mutation in melanoma cells following strong T cell-mediated immune selection. Furthermore, the presence of multiple HLA class I Ag defects within a tumor cell population may reflect the accumulation of multiple escape mechanisms developed by melanoma cells to avoid distinct sequential T cell-mediated selective events.

Mechanism of High Susceptibility of Iron-Overloaded Mouse to Vibrio vulnificus Infection

Vibrio vulnificus produces fulminant septicemia in humans with underlying conditions, particularly those with diseases that elevate the iron level. The effect of a high iron level on the virulence of V. vulnificus was therefore investigated in mice treated with iron dextran. The mice loaded with iron became highly susceptible to V. vulnificus infection, the LD50 (50% lethal dose) decreased five logs when infected per peritoneum. However, when infected via the oral route, the LD50 was affected little unless the mouse was treated with an additional drug such as cyclophosphamide or d‐galactosamine. Mice with or without iron‐overloading died when the bacterial concentration in the blood reached 105 cfu/ml or above. Iron increased the growth rate of the bacteria, both inside and outside of the animal, quickly reaching a lethal concentration in the iron‐overloaded mouse. V. vulnificus, grown with or without the addition of iron, showed strong cytotoxicity on the isolated cells or within the animal at high bacterial concentration. Iron overload stimulated the production of tumor necrosis factor a (TNF‐α), a major factor of septic shock, in mice upon infection with the bacteria, probably caused by the endotoxin; however, the neutrophils, whose migration is effected by TNF‐α, appeared to be less active. Taken together, the major virulence factor of V. vulnificus appeared to be the accelerated growth of bacteria to quickly reach the lethal level and the lower activity of immune cells including neutrophil as a result of iron‐overloading. These two effects manifest other virulence factors, the hosts as well as bacterial. Such factors, other than TNF‐α stimulated by the endotoxin, enhanced cytotoxicity, which kills the host cells including the hosts immune cells.

Defective Human Leukocyte Antigen Class I-associated Antigen Presentation Caused by a Novel β2-Microglobulin Loss-of-function in Melanoma Cells

The major histocompatibility complex class I molecules consist of three subunits, the 45-kDa heavy chain, the 12-kDa β2-microglobulin (β2m), and an ∼8-9-residue antigenic peptide. Without β2m, the major histocompatibility complex class I molecules cannot assemble, thereby abolishing their transport to the cell membrane and the subsequent recognition by antigen-specific T cells. Here we report a case of defective antigen presentation caused by the expression of a β2m with a Cys-to-Trp substitution at position 25 (β2mC25W). This substitution causes misfolding and degradation of β2mC25W but does not result in complete lack of human leukocyte antigen (HLA) class I molecule expression on the surface of melanoma VMM5B cells. Despite HLA class I expression, VMM5B cells are not recognized by HLA class I-restricted, melanoma antigen-specific cytotoxic T lymphocytes even following loading with exogenous peptides or transduction with melanoma antigen-expressing viruses. Lysis of VMM5B cells is restored only following reconstitution with exogenous or endogenous wild-type β2m protein. Together, our results indicate impairment of antigenic peptide presentation because of a dysfunctional β2m and provide a mechanism for the lack of close association between HLA class I expression and susceptibility of tumor cells to cytotoxic T lymphocytes-mediated lysis in malignant diseases.

HLA-G in melanoma: can the current controversies be solved?

The potential role of HLA-G in tumor immune escape has stimulated interest in the analysis of HLA-G antigens in malignant cells. Malignant melanoma is the tumor which has been mostly analyzed for HLA-G expression. Results obtained by seven groups of investigators about HLA-G expression in 108 melanoma cell lines have been concordant. HLA-G mRNA has been found in about 50% of the cell lines tested, whereas HLA-G protein has been found in less than 1% of the cell lines analyzed. In contrast, results obtained from six groups of investigators about HLA-G protein expression in 133 melanoma lesions have been conflicting. The possible causes of these conflicting results as well as the reasons for the discrepancy in HLA-G expression between cultured melanoma cell lines and surgically removed lesions have been discussed. Lastly, data about the potential clinical relevance of HLA-G expression in melanoma has been reviewed. The available data in the literature strongly suggest that progress in this exciting research area would greatly benefit from experiments to solve the current controversies in the field.

Immunotherapy of Melanoma Targeting Human High Molecular Weight Melanoma-Associated Antigen: Potential Role of Nonimmunological Mechanisms

Abstract: Induction of humoral anti‐human high molecular weight melanoma‐associated antigen (anti‐HMW‐MAA) immunity following active specific immunotherapy is associated with a statistically significant prolongation of survival in patients with melanoma. This association does not appear to be mediated by immunological mechanisms because anti‐HMW‐MAA antibodies are poor mediators of complement‐ and cell‐mediated cytotoxicity of melanoma cells. Therefore, we have been investigating nonimmunological mechanisms by which anti‐HMW‐MAA antibodies (Abs) affect the biology of melanoma cells. We have demonstrated that anti‐HMW‐MAA mAbs interfere with the interaction of HMW‐MAA with extracellular matrix (ECM) components, a process known to be crucial in the early phase of melanoma metastasis. Furthermore, anti‐HMW‐MAA mAbs appear to block the series of signal transduction events triggered by the interaction of HMW‐MAA with ECM. They include the activation of the family of Rho GTPases, p130cas, and focal adhesion kinase (FAK). These findings parallel the inhibition of the rat homologue of HMW‐MAA NG2 function by anti‐NG2 antibodies. Taken together, all these results provide a mechanistic explanation not only for the therapeutic effect of anti‐HMW‐MAA antibodies in the treatment of melanoma, but also for the function of HMW‐MAA in the biology of melanoma cells. This information is expected to serve as a useful background to design effective HMW‐MAA‐targeted immunotherapy in patients with melanoma.

HLA Antigen Changes in Malignant Tumors of Mammary Epithelial Origin: Molecular Mechanisms and Clinical Implications

As in other types of malignant tumors in humans and in other animal species, malignant transformation of human mammary epithelial cells may be associated with changes in gene expression and in their antigenic profile. Among the latter, loss or downregulation of classical HLA class I antigens (Fig. 1(A)) and induction of non-classical HLA class I antigens (Fig. 1(B)) as well as of HLA class II antigens (Fig. 1(C)) are of particular interest to tumor immunologists and clinical oncologists because of the critical role these antigens play in the generation of tumor antigen (TA)-specific immune responses [1,2], as well as their ability to modulate the interactions of NK cells [3] and T cell subpopulations [4,5] with target cells (Fig. 1(A)). In this chapter we will first discuss the available information about the frequency of abnormalities in classical HLA class I antigen expression in breast carcinoma lesions and the potential underlying molecular defects. Second, we will address the frequency of the non-classical