Shereen Amria

Gamma-IFN-inducible-lysosomal thiol reductase modulates acidic proteases and HLA class II antigen processing in melanoma

HLA class II-restricted antigen (Ag) processing and presentation are important for the activation of CD4+ T cells, which are the central orchestrating cells of immune responses. The majority of melanoma cells either expresses, or can be induced to express, HLA class II proteins. Thus, they are prime targets for immune mediated elimination by class II-restricted CD4+ T cells. We have previously shown that human melanoma cells lack an important enzyme, gamma interferon-inducible lysosomal thiol-reductase (GILT), capable of perturbing immune recognition of these tumors. Here, we show that GILT expression in human melanoma cells enhances Ag processing and presentation via HLA class II molecules. We also show that GILT expression influences the generation of active forms of cysteinyl proteases, cathepsins B, L and S, as well as an aspartyl protease cathepsin D in melanoma cells. Mechanistic studies revealed that GILT does not regulate acidic cathepsins at the transcriptional level; rather it colocalizes with the cathepsins and influences HLA class II Ag processing. GILT expression in melanoma cells also elevated HLA-DM molecules, which favor epitope loading onto class II in the endolysosomal compartments, enhancing CD4+ T cell recognition. These data suggest that GILT-expressing melanoma cells could prove to be very promising for direct antigen presentation and CD4+ T cell recognition, and may have direct implications for the design of cancer vaccines.

HLA-DM negatively regulates HLA-DR4-restricted collagen pathogenic peptide presentation and T cell recognition

Rheumatoid arthritis, an autoimmune disease, is significantly associated with the HLA class II allele HLA‐DR4. While the etiology of rheumatoid arthritis remains unknown, type II collagen (CII) is a candidate autoantigen. An immunodominant pathogenic epitope from this autoantigen, CII261–273, which binds to HLA‐DR4 and activates CD4+ T cells, has been identified. The non‐classical class II antigen, HLA‐DM, is also a key component of class II antigen presentation pathways influencing peptide presentation by HLA‐DR molecules expressed on professional antigen‐presenting cells (APC). Here, we investigated whether the HLA‐DR4‐restricted presentation of the pathogenic CII261–273 epitope was regulated by HLA‐DM expression in APC. We show that APC lacking HLA‐DM efficiently display the CII261–273 peptide/epitope to activate CD4+ T cells, and that presentation of this peptide is modulated dependent on the level of HLA‐DM expression in APC. Mechanistic studies demonstrated that the CII261–273 peptide is internalized by APC and edited by HLA‐DM molecules in the recycling pathway, inhibiting peptide presentation and T cell recognition. These findings suggest that HLA‐DM expression in APC controls class II‐mediated CII261–273 peptide/epitope presentation and regulates CD4+ T cell responses to this self epitope, thus potentially influencing CII‐dependent autoimmunity.

HLA class II antigen presentation by prostate cancer cells.

Prostate cancer is the second most commonly diagnosed cancer in men. Recent evidence suggests that reduced expression of target protein antigens and human leukocyte antigen (HLA) molecules is the predominant immune escape mechanism of malignant prostate tumor cells. The purpose of this study was to investigate the prospect of antigen specific immunotherapy against prostate cancer via the HLA class II pathway of immune recognition. Here, we show for the first time that prostate cancer cells express HLA class II proteins that are recognized by CD4+ T cells. Prostate tumor cells transduced with class II molecules efficiently presented tumor-associated antigens/peptides to CD4+ T cells. This data suggests that malignant prostate tumors can be targeted via the HLA class II pathway, and that class II-positive tumors could be employed for direct antigen presentation, and CD4+ T-cell mediated tumor immunotherapy.

Defects in HLA class II antigen presentation in B-cell lymphomas

Burkitt Lymphoma (BL) is a high grade B-cell malignancy occurring most frequently in children in areas with holoand hyper-endemic malaria and with lesser frequency in all other parts of the world [1,2]. This neoplasm is one of the fastest growing malignancies in humans and is one of the nonHodgkin lymphomas (NHL) [1 – 3]. Non-Hodgkin Follicular Lymphoma (FL) is also a common type of B-cell lymphoma, comprising approximately 30% of all cases. Transformation of low grade non-Hodgkin lymphomas to high-grade diffuse large B-cell lymphomas are also found in a significant number of patients with B-cell malignancies [4]. Studies have long focused on eliciting tumor-specific CD8þ cytotoxic T-cells (CTL) because of their potent antitumor activity [5]. The resulting treatments, however, have been surprisingly poor at inducing complete tumor rejection. The existence of direct antitumor, effector cytotoxic CD4þ T-cells also remains elusive, although a few studies have shown that CD4þ CTL can directly kill tumor cells [6,7]. By contrast, it is widely believed that helper, HLA class II-restricted non-cytotoxic CD4þ T-cells are important for the induction and maintenance of antitumor immunity exerted by cytotoxic CD8þ CTL. Because the majority of B-cell tumors express HLA class II molecules, these tumors could be potential targets for CD4þ T-cells. Our current study suggests that there is a severe defect in HLA class II antigen (Ag) presentation in B-cell lymphomas. In this letter, we will also show that BL-as well as FL-associated molecules alter the HLA class II pathway for Ag presentation diminishing CD4þ T cell recognition. To examine whether B-cell lymphomas functionally present antigenic peptides to CD4þ T-cells, we analyzed four primary B-cell tumors from B-lymphoma patients. Briefly, lymph node cells and blood samples were obtained from one BL (TB2952) and three FL (TB2759, TB2767 and TB2769) patients through our Hollings Cancer Center Tissue Bank (Medical University of South Carolina, Charleston, USA). TB2952 cells are Burkitt-like lymphoma/ leukemia (EBV status unknown) and positive for CD33, CD10, CD19, CD20 and CD38 as determined by flow cytometric analysis. TB2759 cells are follicular lymphoma/leukemia and positive for CD10, CD19, CD20 and CD52. TB2767 cells are grade 2 FL that expressed CD10, CD19, CD20, CD38, CD45 and CD52. TB2769 cells are grade 3 FL and positive for CD10, CD19, CD20, CD23, CD25, CD38, CD45 and CD52. Blood samples were also obtained from healthy individuals with written consent. Healthy control B-cells and malignant primary tumors (TB2759, TB2767, TB2769 and TB2952) were subjected to western blotting to determine HLA class II protein levels. Western blot analysis showed that these primary B-cells as well as B-cell tumors expressed HLA-DR molecules. To study class II-restricted CD4þ T cell recognition, we transfected these cells with a distinct class II allele HLA-DR4 as described previously [8]. TB2759, TB2767, TB2769 and TB2952 cells expressed measurable surface DR4 molecules as determined

Elevation of c-MYC Disrupts HLA Class II–Mediated Immune Recognition of Human B Cell Tumors

Elevated levels of the transcription factor c-myc are strongly associated with various cancers, and in particular B cell lymphomas. Although many of c-MYC’s functions have been elucidated, its effect on the presentation of Ag through the HLA class II pathway has not been reported previously. This is an issue of considerable importance, given the low immunogenicity of many c-MYC–positive tumors. We report in this paper that increased c-MYC expression has a negative effect on the ability of B cell lymphomas to functionally present Ags/peptides to CD4+ T cells. This defect was associated with alterations in the expression of distinct cofactors as well as interactions of antigenic peptides with class II molecules required for the presentation of class II–peptide complexes and T cell engagement. Using early passage Burkitt’s lymphoma (BL) tumors and transformed cells, we show that compared with B lymphoblasts, BL cells express decreased levels of the class II editor HLA-DM, lysosomal thiol-reductase GILT, and a 47-kDa enolase-like protein. Functional Ag presentation was partially restored in BL cells treated with a c-MYC inhibitor, demonstrating the impact of this oncogene on Ag recognition. This restoration of HLA class II–mediated Ag presentation in early passage BL tumors/cells was linked to enhanced HLA-DM expression and a concurrent decrease in HLA-DO in BL cells. Taken together, these results reveal c-MYC exerts suppressive effects at several critical checkpoints in Ag presentation, which contribute to the immunoevasive properties of BL tumors.

Enhancement of HLA class II-restricted CD4+ T cell recognition of human melanoma cells following treatment with bryostatin-1.

The majority of melanoma cells express detectable levels of HLA class II proteins, and an increased threshold of cell surface class II is crucial for the stimulation of CD4+ T cells. Bryostatin-1, a protein kinase C (PKC) activator, has been considered as a potent chemotherapeutic agent in a variety of in vitro tumor models. Little is known about the role of bryostatin-1 in HLA class II Ag presentation and immune activation in malignant tumors, especially in melanoma. In this study, we show that bryostatin-1 treatment enhances CD4+ T cell recognition of melanoma cells in the context of HLA class II molecules. We also show that bryostatin-1 treatment of melanoma cells increases class II protein levels by upregulating the class II transactivator (CIITA) gene. Flow cytometry and confocal microscopic analyses revealed that bryostatin-1 treatment upregulated the expression of costimulatory molecules (CD80 and CD86) in melanoma cells, which could prolong the interaction of immune cells and tumors. Bryostatin-1 also induced cellular differentiation in melanoma cells, and reduced tumorigenic factors such as pro-cathepsins and matrix-metalloproteinase-9. These data suggest that bryostatin-1 could be used as a chemo-immunotherapeutic agent for reducing tumorigenic potential of melanoma cells while enhancing CD4+ T cell recognition to prevent tumor recurrence.

Disruption of HLA class II antigen presentation in Burkitt lymphoma: implication of a 47 000 MW acid labile protein in CD4+ T-cell recognition

While Burkitt lymphoma (BL) has a well‐known defect in HLA class I‐mediated antigen presentation, the exact role of BL‐associated HLA class II in generating a poor CD4+ T‐cell response remains unresolved. Here, we found that BL cells are deficient in their ability to optimally stimulate CD4+ T cells via the HLA class II pathway. This defect in CD4+ T‐cell recognition was not associated with low levels of co‐stimulatory molecules on BL cells, as addition of external co‐stimulation failed to elicit CD4+ T‐cell activation by BL. Further, the defect was not caused by faulty antigen/class II interaction, because antigenic peptides bound with measurable affinity to BL‐associated class II molecules. Interestingly, functional class II–peptide complexes were formed at acidic pH 5·5, which restored immune recognition. Acidic buffer (pH 5·5) eluate from BL cells contained molecules that impaired class II‐mediated antigen presentation and CD4+ T‐cell recognition. Biochemical analysis showed that these molecules were greater than 30 000 molecular weight in size, and proteinaceous in nature. In addition, BL was found to have decreased expression of a 47 000 molecular weight enolase‐like molecule that enhances class II‐mediated antigen presentation in B cells, macrophages and dendritic cells, but not in BL cells. These findings demonstrate that BL likely has multiple defects in HLA class II‐mediated antigen presentation and immune recognition, which may be exploited for future immunotherapies.

HLA class II defects in Burkitt lymphoma: bryostatin-1-induced 17 kDa protein restores CD4+ T-cell recognition.

While the defects in HLA class I-mediated Ag presentation by Burkitt lymphoma (BL) have been well documented, CD4+ T-cells are also poorly stimulated by HLA class II Ag presentation, and the reasons underlying this defect(s) have not yet been fully resolved. Here, we show that BL cells are deficient in their ability to optimally stimulate CD4+ T cells via the HLA class II pathway. The observed defect was not associated with low levels of BL-expressed costimulatory molecules, as addition of external co-stimulation failed to result in BL-mediated CD4+ T-cell activation. We further demonstrate that BL cells express the components of the class II pathway, and the defect was not caused by faulty Ag/class II interaction, because antigenic peptides bound with measurable affinity to BL-associated class II molecules. Treatment of BL with broystatin-1, a potent modulator of protein kinase C, led to significant improvement of functional class II Ag presentation in BL. The restoration of immune recognition appeared to be linked with an increased expression of a 17 kDa peptidylprolyl-like protein. These results demonstrate the presence of a specific defect in HLA class II-mediated Ag presentation in BL and reveal that treatment with bryostatin-1 could lead to enhanced immunogenicity.

Abstract 4787: GILT regulates antigen processing and CD4+ T cell recognition of melanoma cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Metastatic melanoma is one of the deadliest of skin cancers and is increasing in incidence. Since current treatment regimens are ineffective at curing the disease, novel approaches, such as immunotherapy, for treating this malignant disease are being explored to elicit long-lasting antitumor immunity. We have previously shown that the majority of metastatic melanoma cells express HLA class II molecules, but lack Gamma-Interferon-inducible Lysosomal Thiol reductase (GILT), which differentially regulates HLA class II-restricted antigen processing and presentation. The purpose of this study was to investigate the mechanisms by which GILT insertion influences CD4+ T cells recognition of human melanoma cells via the HLA class II pathway. Biochemical analysis of melanoma cells showed that GILT expression upregulated the HLA class II components such as HLA-DM and invariant chain while class II protein expression remained unchanged. An increase in endolysosomal cathepsin protein expression and activity was also observed in melanoma cells transfected with GILT. Data obtained from biochemical and functional assays suggest that GILT insertion may help melanoma cells generate more antigenic determinants that are readily accessible to class II loading and presentation to CD4+ T cells. Taken together, these data suggest that GILT-inserted melanoma cells could be used in the design of whole cell vaccines for the treatment of metastatic melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4787.