Antigen Processing Machinery Components Research Articles

Upregulation of antigen-processing machinery components at mRNA level in acute lymphoblastic leukemia cells after CD40 stimulation

The development of immunotherapy in hematologic malignancies has been observed in the last few years. One of the approaches is the use of cancer vaccines based on leukemia-derived dendritic cells (DC). Recent studies from our laboratory and other laboratories have shown that CD40 stimulation improves leukemia cells immunogenicity and generates an antitumor immune response. The design of future cancer vaccines requires the knowledge concerning the function of dendritic cells including antigen processing. The aim of our present study was the assessment of antigen-processing machinery (APM) components in acute lymphoblastic leukemia (ALL) cells before and after CD40 stimulation at messenger RNA (mRNA) level. Twenty-five children with ALL were enrolled into the study. Leukemic cells were stimulated (or not) with CD40L and IL-4. Elements of the antigen-processing machinery (MB1, LMP2, LMP7, LMP10, TAP1, TAP2, calnexin, calreticulin, tapasin, ERp57, zeta, delta) were determined by real-time PCR technique. The expression of important costimulatory and adhesion molecules considered as DC markers (CD40, CD54, CD80, CD83, CD86) were determined at the mRNA (PCR) and protein (flow cytometry) levels. The following are the results of our study: (1) We noted an upregulation of all costimulatory and adhesion molecules at the mRNA and protein levels in ALL cells after the culture; (2) the significant rise in expression of nearly all APM components after CD40 stimulation was observed. This confirms specific stimulation of the antigen-processing system in ALL cells by CD40L. Future work should focus on the clinical significance of these findings for immunotherapy in leukemias.

HLA Class I Antigen Down-regulation in Primary Laryngeal Squamous Cell Carcinoma Lesions as a Poor Prognostic Marker

We have investigated the role of antigen-processing machinery (APM) component defects in HLA class I antigen down-regulation in laryngeal squamous cell carcinoma (SCC) lesions and assessed the clinical significance of these defects. To this end, 63 formalin-fixed, paraffin-embedded tumor lesions were examined for APM component and HLA class I antigen expression by immunohistochemistry. Calnexin, calreticulin, and ERp57 were down-regulated in approximately 25% of the lesions tested, whereas LMP2, TAP1, tapasin, and HLA class I antigens were down-regulated in at least 70% of the lesions tested. LMP2 and tapasin expression was significantly correlated with HLA class I antigen expression suggesting APM component defects as a mechanism underlying HLA class I antigen down-regulation in laryngeal SCC lesions. The expression of most APM components and HLA class I antigens was correlated with the extent of CD8+ T cell infiltration into tumor lesions. Furthermore, LMP2 and HLA class I antigen down-regulation and low CD8+ T cell infiltration were significantly associated with reduced patients' survival. Multivariate analysis identified HLA class I antigen down-regulation as an independent unfavorable prognostic marker. This association is likely to reflect the reduction in the extent of CD8+ T cell infiltration in laryngeal SCC lesions.

Coexpression of NRASQ61R and BRAFV600E in Human Melanoma Cells Activates Senescence and Increases Susceptibility to Cell-Mediated Cytotoxicity

Activating mutations in BRAF and NRAS oncogenes in human melanomas are mutually exclusive. This finding has suggested an epistatic relationship but is consistent even with synthetic lethality. To evaluate the latter possibility, a mutated NRAS(Q61R) oncogene was expressed, under a constitutive or a doxycycline-regulated promoter, in a metastatic melanoma clone (clone 21) harboring an activated BRAF(V600E) oncogene. After the first 10 to 12 in vitro passages, the constitutive NRAS(Q61R) transfectant displayed progressive accumulation in G(0)-G(1) phase of the cell cycle and stained for the senescence-associated beta-galactosidase activity (SA-beta-Gal). Inducible expression of NRAS(Q61R), by the Tet-Off system, in clone 21 cells (21NRAS(61ON)) led to overactivation of the RAS/RAF/mitogen-activated protein kinase signaling pathway and, after the 10th in vitro passage, led to promotion of senescence. This was documented by reduced proliferation, flattened cell morphology, reduced growth in Matrigel, positive staining for SA-beta-Gal, and expression of AMP-activated protein kinase and of the cell cycle inhibitor p21(waf1/Cip1). These effects were detected neither in 21 cells with silenced NRAS(Q61R) (21NRAS(61OFF)) nor in cells transfected with an inducible wild-type NRAS gene (21NRAS(WTON)). In addition, when compared with parental 21 cells, or with 21NRAS(61OFF), 21NRAS(61ON) and constitutive NRAS(Q61R) transfectants cells showed increased susceptibility to cytotoxicity by both HLA class I antigen-restricted and nonspecific T cells and up-regulation of several MHC class I antigen processing machinery components. These results suggest a relationship of synthetic lethality between NRAS and BRAF oncogenes, leading to selection against "double-mutant" 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 beta(2)-microglobulin (beta(2)m), and an approximately 8-9-residue antigenic peptide. Without beta(2)m, 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 beta(2)m with a Cys-to-Trp substitution at position 25 (beta(2)m(C25W)). This substitution causes misfolding and degradation of beta(2)m(C25W) 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 beta(2)m protein. Together, our results indicate impairment of antigenic peptide presentation because of a dysfunctional beta(2)m 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.

Association of Antigen-Processing Machinery and HLA Antigen Phenotype of Melanoma Cells with Survival in American Joint Committee on Cancer Stage III and IV Melanoma Patients

Because changes in the expression level of antigen-processing machinery (APM) components and HLA class I and II antigens in melanoma cells are expected to affect their interactions with the immune system of the host, we assessed the clinical relevance of quantitative variations in the expression of these molecules in melanoma lesions. Short-term (<10 in vitro passages) melanoma cell lines isolated from 85 American Joint Committee on Cancer (AJCC) stage III and IV patients were stained with APM component and HLA class I antigen-specific and HLA class II antigen-specific monoclonal antibodies and analyzed by flow cytometry. The phenotype of all tumors was characterized by intertumor and intratumor heterogeneity in the expression of all the markers and by significant correlations in the level of expression of markers belonging to the HLA class I antigen-processing and presentation pathway. Hierarchical clustering of the mean fluorescence intensity data defined two main clusters of tumors. The corresponding groups of patients differed significantly in the overall survival but not in other relevant clinical variables, including AJCC stage and therapy received after surgery. Cox regression analysis showed that beta2-microglobulin and HLA class II antigen expression were significantly associated with patients' survival. This evidence was corroborated by the immunohistochemical analysis for HLA class II antigen expression of melanoma lesions from an unrelated group of 52 AJCC stage III and IV patients. These results suggest that quantitative variations in APM component and HLA expression in melanoma lesions from AJCC stage III and IV patients may have an effect on the clinical course of the disease.

Frequent Loss of HLA-A2 Expression in Metastasizing Ovarian Carcinomas Associated with Genomic Haplotype Loss and HLA-A2-Restricted HER-2/neu-Specific Immunity

Defective expression of HLA class I molecules is common in tumor cells and may allow escape from CTL-mediated immunity. We here investigate alterations in expression of HLA class I and their underlying molecular mechanisms in ovarian cancer patients. The HLA class I and HLA-A2 expression levels on noncultured tumor cells of 12 patients diagnosed with ovarian carcinoma were investigated by flow cytometry. Molecular analyses of antigen-processing machinery (APM) components were done in metastatic cancer cells, and the HLA genotype was determined in both these and the primary tumor. HER-2/neu-specific immunity was evaluated by enzyme-linked immunospot assays. The metastatic tumor cells from all patients expressed low levels of HLA class I surface antigens. In six of nine HLA-A2+ patients, HLA-A2 expression was heterogeneous with a subpopulation of tumor cells exhibiting decreased or absent HLA-A2 expression. One patient-derived tumor cell line completely lacked HLA-A2 but exhibited constitutive expression of APM components and high HLA class I expression that was further inducible by IFN-gamma treatment. Genotyping showed a haplotype loss in the metastatic tumor cells, whereas tumor tissue microdissected from the primary tumor exhibited an intact HLA gene complex. Interestingly, HLA-A2-restricted HER-2/neu-specific T-cell responses were evident among the lymphocytes of this patient. Abnormalities in HLA class I antigen expression are common features during the progression of ovarian cancer, and haplotype loss was, for the first time, described as an underlying mechanism.

Human Leukocyte Antigen and Antigen Processing Machinery Component Defects in Astrocytic Tumors

To determine the frequency of abnormalities in human leukocyte antigen (HLA) and antigen processing machinery (APM) component expression in malignant brain tumors. This information may contribute to our understanding of the immune escape mechanisms used by malignant brain tumors because HLA antigens mediate interactions of tumor cells with the host's immune system. Eighty-eight surgically removed malignant astrocytic tumors, classified according to the WHO criteria, were stained in immunoperoxidase reactions with monoclonal antibody recognizing monomorphic, locus-specific, and allospecific determinants of HLA class I antigens, beta2-microglobulin, APM components (LMP2, LMP7, TAP1, TAP2, calnexin, calreticulin, and tapasin), and HLA class II antigens. HLA class I antigens were lost in approximately 50% of the 47 glioblastoma multiforme (GBM) lesions and in approximately 20% of the 18 grade 2 astrocytoma lesions stained. Selective HLA-A2 antigen loss was observed in approximately 80% of the 24 GBM lesions and in approximately 50% of the 12 grade 2 astrocytoma lesions stained. HLA class I antigen loss was significantly (P < 0.025) correlated with tumor grade. Among the APM components investigated, tapasin expression was down-regulated in approximately 20% of the GBM lesions analyzed; it was associated, although not significantly, with HLA class I antigen down-regulation and tumor grade. HLA class II antigen expression was detected in approximately 30% of the 44 lesions analyzed. The presence of HLA antigen defects in malignant brain tumors may provide an explanation for the relatively poor clinical response rates observed in the majority of the T cell-based immunotherapy clinical trials conducted to date in patients with malignant brain tumors.

Antitumor Activity of Human Papillomavirus Type 16 E7–Specific T Cells against Virally Infected Squamous Cell Carcinoma of the Head and Neck

Human papillomavirus (HPV)-associated squamous cell carcinoma of the head and neck (SCCHN) seems to be a suitable target for cancer vaccination. HPV-encoded oncogenic proteins, such as E7, are promising tumor-specific antigens and are obligatory for tumor growth. Because few immunologic studies have analyzed the endogenous HPV-specific immune response in this subset of SCCHN patients, we studied T-cell frequencies against HPV-16 E7(11-20) or E7(86-93) in tumor-bearing, human leukocyte antigen (HLA)-A*0201+ SCCHN patients, whose tumors were either HPV-16+ or HPV-16-. In HPV-16+ SCCHN patients, frequencies of T cells against either peptide were significantly elevated (P < 0.005) compared with HPV-16- patients or healthy volunteers. Tetramer+ T cells showed evidence of terminally differentiated phenotype (CD45RA+CCR7-) and an elevated level of CD107a staining for degranulation. Despite detectable expression of the restricting HLA class I allele, HLA-A*0201-E7(11-20)- or HLA-A*0201-E7(86-93)-specific CTL obtained by in vitro stimulation of healthy donor peripheral blood mononuclear cells only recognize a naturally HPV-16-transformed, HLA-A*0201+ SCCHN cell line after pretreatment with IFN-gamma. This cell line had little or no expression of LMP2, TAP1, and tapasin, critical components of the HLA class I antigen-processing machinery, which were up-regulated by IFN-gamma treatment. Immunohistochemistry of HPV-16+ SCCHN tumors showed that these antigen-processing machinery components are down-regulated in tumors in vivo compared with adjacent normal squamous epithelium. Thus, immunity to HPV-16 E7 is associated with the presence of HPV-16 infection and presentation of E7-derived peptides on SCCHN cells, which show evidence of immune escape. These findings support further development of E7-specific immunotherapy and strategies for up-regulation of antigen-processing machinery components in HPV-associated SCCHN.

Immunoselective Pressure and Human Leukocyte Antigen Class I Antigen Machinery Defects in Microsatellite Unstable Colorectal Cancers

In colorectal cancer, the immune response is particularly pronounced against tumors displaying the high microsatellite instability (MSI-H) phenotype. MSI-H tumors accumulate mutations affecting microsatellites located within protein encoding regions (coding microsatellites, cMS), which lead to translational shifts of the respective reading frames. Consequently, novel tumor-specific frameshift-derived neopeptides (FSP) are generated and presented by MSI-H tumor cells, thus eliciting effective cytotoxic immune responses. To analyze whether the immunoselective pressure was reflected by the phenotype of MSI-H colorectal cancer cells, we compared here the expression of antigen processing machinery (APM) components and human leukocyte antigen (HLA) class I antigen subunits in 20 MSI-H and 20 microsatellite-stable (MSS) colorectal cancer using a panel of newly developed APM component-specific monoclonal antibodies. In addition, we did a systematic analysis of mutations at cMS located within APM genes and beta2-microglobulin (beta2m). Total HLA class I antigen loss was observed in 12 (60.0%) of the 20 MSI-H lesions compared with only 6 (30.0%) of the 20 MSS colorectal cancer lesions. Moreover, total loss of membraneous HLA-A staining was significantly more frequent in MSI-H colorectal cancer (P = 0.0024). Mutations at cMS of beta2m and genes encoding APM components (TAP1 and TAP2) were detected in at least 7 (35.0%) of 20 MSI-H colorectal cancers but in none of the MSS colorectal cancers (P = 0.0002). These data show that defects of HLA class I antigen processing and presentation seem to be significantly more frequent in MSI-H than in MSS colorectal cancer, suggesting that in MSI-H colorectal cancer the immunoselective pressure leads to the outgrowth of cells with defects of antigen presentation.

T-Cell activation by t(9;22) acute lymphoblastic leukemia-derived dendritic-like cells is associated with increased tapasin expression.

Dendritic-like cells from t(9;22) acute lymphoblastic leukemia (ALL) blasts can activate T cells, while the original unmodified leukemic blasts cannot. To determine whether these functional differences were associated with differences in antigen-processing machinery (APM) component expression, we have measured the level of APM component expression in unmodified blasts and ALL-derived dendritic-like cells. Seven t(9;22) ALL patient samples and one cell line were studied for APM component expression utilizing a unique panel of recently developed monoclonal antibodies and a recently developed intracellular staining technique. In addition, the HLA class I antigen cell surface expression was measured. HLA class I antigens were similarly expressed on the unmodified blasts and on the autologous dendritic-like cells. Intracellular HLA class I antigen and tapasin expression (P=0.03 for both) were upregulated in all t(9;22) ALL-derived dendritic-like cells, in comparison to the unmodified blasts. These results provide a potential mechanism for the ability of t(9;22) ALL-derived dendritic-like cells to induce T-cell activation and, suggest that tapasin upregulation may serve as a marker to standardize and monitor the quality of the dendritic-like cells used in immunotherapy.

Induction of HLA-G expression in a melanoma cell line OCM-1A following the treatment with 5-aza-2′-deoxycytidine

The non-classical HLA class I antigen HLA-G is an immune modulator which inhibits the functions of T cells, NK cells, and the Dendritic cells (DC). As a result, HLA-G expression in malignant cells may provide them with a mechanism to escape the immune surveillance. In melanoma, HLA-G antigen expression has been found in 30% of surgically removed lesions but in less than 1% of established cell lines. One possible mechanism underlying the differential HLA-G expression in vivo and in vitro is that the HLA-G gene is epigenetically repressed in melanoma cells in vitro. To test this hypothesis, we treated the HLA-G negative melanoma cell line OCM-1A with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-AC) and analyzed whether HLA-G expression can be restored. Our data strongly suggest that HLA-G is silenced as a result of CpG hypermethylation within a 5' regulatory region encompassing 220 bp upstream of the start codon. After treatment, HLA-G mRNA expression was dramatically increased. Western blot and flow cytometry showed that HLA-G protein was induced. Interestingly, HLA-G cell surface expression on the 5-AC treated OCM-1A cells is much less than that on the HLA-G positive JEG-3 cells while a similar amount of total HLA-G was observed. Possible mechanisms for the difference were analyzed in the study such as cell cold-treatment, peptide loading and antigen processing machinery components (APM) as well as beta2 microglobulin (beta2-m) expression. Data revealed that the APM component calreticulin might be involved in the lower HLA-G surface expression on OCM-1A cells. Taken together, our results indicated that DNA methylation is an important epigenetic mechanism by which HLA-G antigen expression is modulated in melanoma cells in vitro. Furthermore, to the first time, we hypothesized that the deficiency of calreticulin might be involved in the low HLA-G surface expression on the 5-AC treated OCM-1A cells.

Mechanisms of immune evasion of human neuroblastoma

Neuroblastoma (NB) is a common neuroectodermal tumor of childhood with low response to conventional therapy in patients with advanced stage disease. Among novel strategies, immumotherapy has attracted much interest. However, scanty information is available about the immunogenicity of human NB. Here, we review our data showing that human NB may evade the control mediated by T cytotoxic lymphocytes and natural killer (NK) cells through multiple mechanisms: (i) downregulation of HLA class I molecules and antigen processing machinery components (ii) downregulation of activating ligands for the activating immunoreceptor NKG2D expressed by cytotoxic T lymphocytes and NK cells. Additional mechanisms of immune evasion used by human NB cells are discussed.

Multiple defects of the antigen-processing machinery components in human neuroblastoma: immunotherapeutic implications

Low expression of human leukocyte antigen (HLA) class I in human tumors may be related to defects of the antigen-processing machinery (APM) components. Neuroblastoma cells are virtually HLA class I negative, but (i) the underlying mechanisms are unknown, and (ii) expression of the APM components has never been investigated. Here we have used a panel of novel monoclonal antibodies to proteasomal and immunoproteasomal components, chaperons and transporter associated with antigen processing (TAP) to characterize 24 stroma-poor neuroblastoma tumors and six neuroblastoma cell lines. Primary tumors showed defects in the expression of zeta, tapasin, TAP1 or TAP2, HLA class I heavy chain and beta2 microglobulin, LMP2 and LMP7, as compared to normal adrenal medulla. Neuroblastoma cell lines displayed roughly similar patterns of APM expression in comparison to primary tumors. Incubation of neuroblastoma cell lines with interferon-gamma caused upregulation of HLA class I molecules and reduced lysis by killer inhibitory receptor HLA ligand-matched NK cells. Defects in APM components explain reduced peptide loading on HLA class I molecules, their instability and failure to be expressed on the cell surface. HLA class I upregulation by interferon-gamma, although enhancing neuroblastoma cell recognition by cytotoxic T cells, dampens their susceptibility to NK cells.

A method to generate antigen-specific mAb capable of staining formalin-fixed, paraffin-embedded tissue sections

Abnormalities in HLA class I antigen expression are frequently found in malignant tumors. Their potential role in the clinical course of the disease and in the outcome of T cell-based immunotherapy has stimulated interest in the characterization of the molecular mechanisms underlying HLA class I antigen abnormalities in malignant cells. Multiple mechanisms have been identified. Among them are abnormalities in antigen processing machinery (APM) component expression. In spite of this information, APM component expression in malignant lesions has been investigated only to a limited extent because of the lack of availability, for most APM components, of monoclonal antibodies (mAb) which stain formalin-fixed, paraffin-embedded tissues. The latter are the substrate of choice in immunohistochemical (IHC) reactions. To overcome this limitation, we have developed a simple and reproducible method to generate APM component-specific mAb which stain formalin-fixed, paraffin-embedded tissue sections. This method involves five steps: (i) immunogenic amino acid sequences, which display low homology with their mouse counterparts when possible, are identified in APM components and utilized to synthesize peptides; (ii) BALB/c mice are immunized with keyhole limpet hemocyanin (KLH)-conjugated synthetic peptides and with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-purified recombinant APM component proteins; (iii) immunized mice, which develop high titer APM component-specific antibodies, are utilized to generate hybridomas which are screened for APM component-specific antibody production by Western blotting assays, with lymphoid cell lysates; (iv) identified APM component-specific mAb are characterized in their specificity and in their reactivity with permeabilized cells in ELISA and/or flow cytometry; and (v) mAb, with the appropriate reactivity pattern, are tested in IHC reactions with formalin-fixed, paraffin-embedded tissue sections. The use of the methodology we have developed resulted in the generation of a panel of APM component-specific mAb capable of staining formalin-fixed, paraffin-embedded tissue sections in IHC reactions. These reagents will facilitate the analysis of APM component expression in tissues under physiological and pathological conditions. In addition, the methodology we have described is likely to be applicable to other antigenic systems to develop mAb capable of detecting protein components of interest in formalin-fixed, paraffin-embedded tissue sections.

Defects in the Human Leukocyte Antigen Class I Antigen Processing Machinery in Head and Neck Squamous Cell Carcinoma: Association with Clinical Outcome

Human leukocyte antigen (HLA) class I antigen defects, which are frequently present in head and neck squamous cell carcinoma (HNSCC) cells may provide the tumor with an escape mechanism from immune surveillance. Scanty information is available about mechanisms underlying HLA class I antigen defects in both lesions and cell lines from HNSCC. In this study, we investigate the role of antigen processing machinery (APM) component abnormalities in the generation of deficient HLA class I surface expression of HNSCC cells. Using immunohistochemistry, Western blot, and RT-PCR analyses we correlated the expression of the IFN-gamma inducible proteasome subunits and of the peptide transporter TAP with that of HLA class I antigens in biopsies and cell lines from primary, recurrent, and metastatic HNSCC. Furthermore, APM component and HLA class I antigen expression in surgically removed lesions were correlated with the course of the disease in order to assess the clinical significance of deficient expression of these molecules. A high frequency of LMP2, LMP7, and TAP1 down-regulation or loss was found in tumor lesions and cell lines obtained from HNSCC cancer patients. These defects could be corrected by incubating cells with IFN-gamma. Furthermore, LMP2, LMP7, TAP1, TAP2, and HLA class I antigen expression rates in primary HNSCC lesions were found to predict overall survival. Lastly, the level of LMP7 expression was significantly associated with disease recurrence at 2 years. Our results suggest that the analysis of APM component expression in HNSCC lesions can provide useful prognostic information in patients with HNSCC.

Immune selection of hot-spot beta 2-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 beta(2)-microglobulin (beta(2)m) 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 beta(2)m 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 beta(2)m 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.

Human Leukocyte Antigen (HLA) Class I Defects in Head and Neck Cancer: Molecular Mechanisms and Clinical Significance

The potential role of immunological events in the pathogenesis and clinical course of head and neck squamous cell carcinoma (SCCHN) has stimulated interest in the characterization of HLA class I antigen expression in SCCHN lesions, because these molecules play an important role in the interaction of malignant cells with the host's immune system. Therefore in this paper, following a description of the methodology used to analyze HLA class I antigen expression in normal tissues and in malignant lesions, we have reviewed data about the frequency of HLA class I antigen defects in about 500 primary and in about 25 metastatic SCCHN lesions. The mean frequency of total HLA class I antigen loss in primary and metastatic lesions is approx 15% and 40%, respectively. The mean frequency of selective HLA class I antigen loss in primary lesions is approx 37%. This type of abnormality has not been investigated in metastatic lesions so far. The molecular mechanisms underlying HLA class I antigen defects in SCCHN cells have been investigated to a limited extent. The available information suggests that structural defects in beta2m genes are rare. In contrast, functional abnormalities of the antigen processing machinery (APM) components are frequent in SCCHN cells. The latter abnormalities are likely to account for the unusual finding that most of SCCHN cell lines are resistant in vitro to HLA class I antigen restricted, tumor antigen (TA)-specific CTL recognition under basal conditions in spite of the expression of TA and HLA class I antigens. CTL recognition of SCCHN cells is restored by incubation with IFN-gamma. These in vitro findings provide a mechanism for the association between APM component defects in SCCHN lesions and clinical course of the disease and imply that T cell-based immunotherapy of SCCHN may benefit from the intralesional administration of IFN-gamma.

T Cell Activation by t(9;22) Acute Lymphoblastic Leukemia-Derived Dendritic-Like Cells Is Associated with Increased Expression of Antigen Processing Machinery Components.

We have recently shown that dendritic-like cells derived from t(9;22) acute lymphoblastic leukemia (ALL) cells can activate T cells, while the original unmodified leukemic blasts cannot. To define the molecular basis of this functional difference, we have compared the level of antigen processing machinery components in unmodified blasts and ALL-derived dendritic-like cells, since they play a major role in antigen presentation. Six patient samples and one t(9;22) ALL cell line (Z119) were studied.Dendritic-like cells were generated by in vitro culture with CD40 ligand, interleukin (IL)-1β, IL-3, IL-7, stem cell factor and tumor necrosis factor-α for six days. Maturation was verified by morphology and CD80/CD86 expression. Cells were studied for HLA class I and class II antigen expression. Furthermore, antigen processing machinery component expression was analyzed by intracellular staining with a unique panel of monoclonal antibodies which recognize the constitutive proteasome (Z, MB1, delta) and immunoproteasome (LMP2, LMP7, LMP10) subunits, the transporter subunits TAP 1 and 2 and the chaperones calnexin, ERp57, calreticulin and tapasin. HLA Class I antigen and HLA-DR/DQ/DP antigen expression was significantly up-regulated on the dendritic-like cells. LMP2, LMP7 and tapasin expression was significantly up-regulated in all t(9;22) ALL-derived dendritic-like cells, in comparison to the unmodified blasts. No significant difference was detected in the expression of the other antigen processing machinery components. These results suggest that T cell activation by t(9;22) ALL-derived dendritic-like cells is associated with increased expression of some, but not all, of the antigen processing machinery components. Whether t(9;22) ALL-derived dendritic-like cells can be used as a cellular vaccine for adoptive immunotherapy of ALL is being investigated.

Evolutionary and functional perspectives of the major histocompatibility complex class I antigen-processing machinery

Major histocompatibility complex (MHC) class I molecules present antigenic peptides to CD8+ T cells, providing the basis for immune recognition of pathogen-infected cells. Peptides generated mainly by proteasomes in the cytosol are transported into the lumen of the endoplasmic reticulum by transporters associated with antigen processing (TAP). The maturation of MHC class I molecules is controlled by a number of accessory proteins and chaperones that are to a varying degree dedicated to the assembly of MHC class I. Several newly characterised proteins have been demonstrated to play important roles in this process. This review focuses on the functional relationship and evolutionary history of the antigen-processing machinery (APM) components and MHC class I itself. These are of great interest for further elucidating the origin of the immune system and understanding the mechanisms of antigen presentation and immunology in general.

HER-2/neu-Mediated Regulation of Components of the MHC Class I Antigen-Processing Pathway

Because of its amplification and/or overexpression in many human tumors, the HER-2/neu proto-oncogene represents an attractive target for T-cell-mediated vaccination strategies. However, overexpression of oncogenes is often associated with defective expression of components of the MHC class I antigen-processing machinery (APM), thereby resulting in an immune escape phenotype of oncogene-transformed cells. To determine whether HER-2/neu influences the MHC class I antigen-processing pathway, the expression pattern of different APM components was examined in murine in vitro models of constitutive and tetracycline-controlled HER-2/neu expression. In comparison with HER-2/neu(-) control cells, HER-2/neu(+) fibroblasts exhibit reduced levels of MHC class I surface antigens that were associated with impaired expression and/or function of the peptide transporter associated with antigen processing, the proteasome subunits low molecular weight protein 2 and low molecular weight protein 10, the proteasome activators PA28alpha and PA28beta, and tapasin. These APM abnormalities resulted in reduced sensitivity to lysis by CTLs. The HER-2/neu-mediated immune escape phenotype could be corrected by IFN-gamma treatment. The clinical relevance of this finding was supported by an inverse correlation between HER-2/neu and the peptide transporter associated with antigen-processing protein expression as determined by immunhistochemical analysis of a series of HER-2/neu(-) and HER-2/neu(+) breast cancer specimens. Thus, a functional link between deficient APM component expression and HER-2/neu overexpression is proposed that might influence the design of HER-2/neu-targeted T-cell-based immunotherapeutic strategies.