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.

Glioblastoma is the most common and highly malignant brain tumor. It is also one among the most therapy-resistant human neoplasias. Patients die within a year of diagnosis despite the use of available treatment strategies such as surgery, radiotherapy, and chemotherapy. Thus, there is a critical need to find a novel therapeutic strategy for treating this disease. Here, we have investigated the molecular mechanisms for induction of apoptosis as well as for activation of immune components in human malignant glioblastoma T98G and U87MG cells following treatment with all-trans retinoic acid (ATRA) plus interferon-gamma (IFN-gamma). Treatment of glioblastoma cells with ATRA alone prevented cell proliferation and induced astrocytic differentiation, while IFN-gamma alone induced apoptosis and modulated expression of human leukocyte antigen (HLA) class II molecules such as HLA-DRalpha, HLA-DR complex, invariant chain (Ii), HLA-DM (an important catalyst of the class II-peptide loading), and gamma interferon-inducible lysosomal thiol-reductase (GILT). Interestingly, both T98G and U87MG cells showed more increase in apoptosis with expression of the HLA class II components for an effective immune response following treatment with ATRA plus IFN-gamma than with IFN-gamma alone. Apoptotic mode of cell death was confirmed morphologically by Wright staining and biochemically by measuring an increase in caspase-3 activity. While conversion of tumor cells into HLA class II+/Ii- cells by stimulation with the helper CD4+ T cells is thought to be challenging, this study reports for the first time that treatment of glioblastoma cells with ATRA plus IFN-gamma can simultaneously enhance apoptosis and expression of the HLA class II immune components with a marked suppression of Ii expression. Taken together, this study suggests that induction of apoptosis and immune components of the HLA class II pathway by ATRA plus IFN-gamma may be a promising chemoimmunotherapeutic strategy for treatment of human malignant glioblastoma.

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, CII [261][262][263][264][265][266][267][268][269][270][271][272][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 CII 261-273 epitope was regulated by HLA-DM expression in APC. We show that APC lacking HLA-DM efficiently display the CII [261][262][263][264][265][266][267][268][269][270][271][272][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 CII [261][262][263][264][265][266][267][268][269][270][271][272][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 IImediated CII [261][262][263][264][265][266][267][268][269][270][271][272][273] peptide/epitope presentation and regulates CD4 + T cell responses to this self epitope, thus potentially influencing CII-dependent autoimmunity.Key words: CD4 + T cells Á HLA-DM Á HLA-DR Á Peptide presentation Á Type II collagen IntroductionType II collagen (CII) is the major protein component of articular cartilage [1]. It is thought that T and B cell responses to this autoantigen are associated with the development and pathogenesis of rheumatoid arthritis (RA) [2,3]. It is also widely believed that RA is genetically linked to HLA class II molecules, including some HLA-DR4 alleles, and that T cell responses in collagendependent RA are directed towards the immunodominant pathogenic epitope CII [261][262][263][264][265][266][267][268][269][270][271][272][273] [4,5]. Studies also suggest that T cells from patients with RA predominantly recognize the glycosylated form of the immunodominant CII peptide [6,7]. Professional antigen-presenting cells (APC) such as B cells, macrophages and dendritic cells abundantly express HLA class II proteins and play critical roles in the pathogenesis of autoimmune arthritis by presenting autoantigens to T cells [4,5,8].In RA, cartilage proteins including CII undergo degradation by proteases, leading to the generation of peptides that can bind to HLA class II proteins and trigger CD4 + T cell activation [9]. Studies suggest that the peptides are loaded onto class II proteins Eur. J. Immunol. 2008. 38: 1961-1970 Immunomodulation in the endosomal and lysosomal compartments of APC prior to transit to the cell surface for T cell recognition [5...