Studies aimed at elucidating the immunological and prognostic significance of HLA-DR expression on breast carcinoma cells have yielded contradictory results. To expand on previous studies, we have investigated the associations of tumor cell expression of HLA-DR and its related co-chaperones, invariant chain (Ii) and HLA-DM, with infiltrating inflammatory cells, in situ cytokine mRNA levels and prognosis and outcome in 112 breast carcinoma patients with a median follow-up of 59 months. While the majority of HLA-DR+ tumors co-express Ii, only a minority express HLA-DM. Tumor cell expression of HLA-DR and co-chaperones positively associated with both infiltrating CD4+ and CD8+ T-cell subsets (P < 0.01). Expression of HLA-DR and Ii associated with decreased estrogen receptor alpha levels and younger age at diagnosis, suggesting a role for hormones in the control of HLA class II expression in breast carcinoma. Patients with DR+Ii+DM- tumors had markedly decreased recurrence-free and disease-specific survival as compared with patients with DR+Ii+DM+ tumors (P < 0.05) and HLA-DR/co-chaperone expression was an independent predictor of survival by multivariate Cox regression analysis, controlling for standard prognostic indicators. Tumors that co-express HLA-DR, Ii and HLA-DM have increased levels of IFN-gamma, IL-2 and IL-12 mRNA, suggesting improved survival of patients with DR+Ii+DM+ tumors may be attributable to Th1-dominated immunity. We conclude that expression of determinants of the immune response by tumor cells may influence breast tumor progression and patient outcome.

The coordinate regulation of HLA class II (HLA-II) is controlled by the class II transactivator, CIITA, and is crucial for the development of anti-tumor immunity. HLA-II in breast carcinoma is associated with increased IFN-γ levels, reduced expression of the estrogen receptor (ER) and reduced age at diagnosis. Here, we tested the hypothesis that estradiol (E2) and ERα signaling contribute to the regulation of IFN-γ inducible HLA-II in breast cancer cells. Using a panel of established ER− and ER+ breast cancer cell lines, we showed that E2 attenuated HLA-DR in two ER+ lines (MCF-7 and BT-474), but not in T47D, while it augmented expression in ER− lines, SK-BR-3 and MDA-MB-231. To further study the mechanism(s), we used paired transfectants: ERα+ MC2 (MDA-MB-231 c10A transfected with the wild type ERα gene) and ERα− VC5 (MDA-MB-231 c10A transfected with the empty vector), treated or not with E2 and IFN-γ. HLA-II and CIITA were severely reduced in MC2 compared to VC5 and were further exacerbated by E2 treatment. Reduced expression occurred at the level of the IFN-γ inducible CIITA promoter IV. The anti-estrogen ICI 182,780 and gene silencing with ESR1 siRNA reversed the E2 inhibitory effects, signifying an antagonistic role for activated ERα on CIITA pIV activity. Moreover, STAT1 signaling, necessary for CIITA pIV activation, and selected STAT1 regulated genes were variably downregulated by E2 in transfected and endogenous ERα positive breast cancer cells, whereas STAT1 signaling was noticeably augmented in ERα− breast cancer cells. Collectively, these results imply immune escape mechanisms in ERα+ breast cancer may be facilitated through an ERα suppressive mechanism on IFN-γ signaling.

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SummaryThe human immunoddiciency, type II bare lymphocyte syndrome (BLS), has been attributed to a defect in the transcription of class II histocompatibility genes. Immunocompetence~ as assessed by functional exogenous antigen presentation, was not restored in immortalized B cells, derived from a BLS patient, after transfection with HLA-DR class II structural genes. Incubation of protein antigens, as well as infectious virus, with DR-transfected BLS cells failed to induce activation of antigen-specific helper T lymphocytes. Peptide antigens were presented by class II molecules displayed on BLS cells, although the conformation of these class II proteins was altered as indicated by epitope mapping. This defect in antigen presentation was independent of the specific class II DR allele transfected into BLS cells. Genetic complementation analysis has been used with BLS cells to demonstrate that the defect in class II gene transcription is linked to the absence of a tram-acting factor. Similarly, functional class II dimers were restored after in vitro fusion of cells derived from two distinct BLS complementation groups, implying that specific transcriptional control elements are shared by a gene critical for antigen presentation and genes encoding HLA class II antigens. Thus, two important functionally linked pathways of class II molecules, structural gene expression and antigen presentation, share a common regulatory pathway defective in BLS.T ype II bare lymphocyte syndrome (BLS) or HLA class II-ddicient combined immunodeficiency is an autosomal recessive disorder in which HLA class II structural genes are not transcribed due to a mutation in a gene encoded outside of the MHC (1). This lack of HLA class II expression compromises patient immunity, leading to recurrent bacterial and viral infections. Several lines of evidence suggest that the BLS phenotype results from a defect in a trans-acting factor necessary for transcription of the coordinately regulated class II genes (reviewed in reference 2).Clustering of genes for histocompatibility antigens and accessory molecules required for antigen presentation may ensure the coordinate expression and function of these molecules. The HLA class I and class II structural genes on chromosome 6 are interspersed with genes necessary for antigen responsiveness (3). The ability of class I molecules to present antigens is dependent upon functional transporters associated with antigen processing (TAP) genes which are required to supply peptides to class I molecules in the endoplasmic reticulum (reviewed in reference 3). The TAP genes map between the HLA-DQ and -DP structural genes; in addition to this genetic proximity, they are coregulated with class Iot structural genes by IFN-7 (3). Similarly, genes essential for appropriate class II-mediated antigen presentation map within this same region of the MHC (4, 5). Transcriptional regulation of genes encoded outside the MHC may also be coordinated with histocompatibility antigens to facilitate immune function. Class II antigens and t...

Many human solid tumors express MHC class II (MHC-II) molecules, and proteins normally localized to melanosomes give rise to MHC-II-restricted epitopes in melanoma. However, the pathways by which this response occurs have not been defined. We analyzed the processing of one such epitope, gp10044–59, derived from gp100/Pmel17. In melanomas that have down-regulated components of the melanosomal pathway, but constitutively express HLA-DR*0401, the majority of gp100 is sorted to LAMP-1high/MHC-II+ late endosomes. Using mutant gp100 molecules with altered intracellular trafficking, we demonstrate that endosomal localization is necessary for gp10044–59 presentation. By depletion of the AP-2 adaptor protein using small interfering RNA, we demonstrate that gp100 protein internalized from the plasma membrane to such endosomes is a major source for gp10044–59 epitope production. The gp100 trapped in early endosomes gives rise to epitopes that are indistinguishable from those produced in late endosomes but their production is less sensitive to inhibition of lysosomal proteases. In melanomas containing melanosomes, gp100 is underrepresented in late endosomes, and accumulates in stage II melanosomes devoid of MHC-II molecules. The gp10044–59 presentation is dramatically reduced, and processing occurs entirely in early endosomes or stage I melanosomes. This occurrence suggests that melanosomes are inefficient Ag-processing compartments. Thus, melanoma de-differentiation may be accompanied by increased presentation of MHC-II restricted epitopes from gp100 and other melanosome-localized proteins, leading to enhanced immune recognition.