Upregulation of HLA Expression in Primary Uveal Melanoma by Infiltrating Leukocytes

Abstract

Introduction: Uveal melanoma (UM) with an inflammatory phenotype, characterized by infiltrating leukocytes and increased human leukocyte antigen (HLA) expression, carry an increased risk of death due to metastases. These tumors should be ideal for T-cell based therapies, yet it is not clear why prognostically-infaust tumors have a high HLA expression. We set out to determine whether the level of HLA molecules in UM is associated with other genetic factors, HLA transcriptional regulators, or microenvironmental factors.

Methods: 28 enucleated UM were used to study HLA class I and II expression, and several regulators of HLA by immunohistochemistry, PCR microarray, qPCR and chromosome SNP-array. Fresh tumor samples of eight primary UM and four metastases were compared to their corresponding xenograft in SCID mice, using a PCR microarray and SNP array.

Results: Increased expression levels of HLA class I and II showed no dosage effect of chromosome 6p, but, as expected, were associated with monosomy of chromosome 3. Increased HLA class I and II protein levels were positively associated with their gene expression and with raised levels of the peptide-loading gene TAP1, and HLA transcriptional regulators IRF1, IRF8, CIITA, and NLRC5, revealing a higher transcriptional activity in prognostically-bad tumors. Implantation of fresh human tumor samples into SCID mice led to a loss of infiltrating leukocytes, and to a decreased expression of HLA class I and II genes, and their regulators.

Conclusion: Our data provides evidence for a proper functioning HLA regulatory system in UM, offering a target for T-cell based therapies.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Chromosomal aberrations and HLA expression.

Comparison between chromosomal aberrations and the expression of HLA class I and II antigens in a set of 27 primary UM. Tumors are divided according to their chromosome 3 and 6 status (disomy or monosomy of chromosome 3, and disomy of chromosome 6 or gain of 6p). HLA gene expression was determined using an Illumina microarray (A) and protein expression by immunohistochemistry (B) in UM. Additionally, HLA gene expression was determined using qPCR, which served to validate the Illumina findings (C). Four data points of the qPCR that are outside the axis limits (> 11 and

Fig 2. Tumor infiltrating immune cells.

Association between a low or high density of tumor-infiltrating CD3+ (A), and CD68+ (B)cells and several HLA and HLA-related genes (Illumina array) in primary UM. CD3 (cells/mm2) and CD68 (pixels x 103/mm2) scores were dichotomized at the median.

Fig 3. Effect of the absence of human leukocytes.

Gene-expression (log 2 intensity values) of HLA-B, HLA-DRA, CD3D (as marker for T-cells), and CD163 (as marker for macrophages), of the original tumors (patient) compared to the xenografts (xeno). MP’s are primary tumors; MM’s are metasisis.

Fig 4. Schematic illustration of tumor characteristics and infiltrate.

UM with monosomy 3 attract an infiltrate, producing different cytokines, including Interferon-gamma. The tumor cell (UM cell) responds by increasing HLA class I and II levels, as well as rendering the infiltrating immune cells ineffective (immune suppression) and creating a tumor-favorable environment, with amongst others, stimulation of angiogenesis.