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Mechanisms and Effects of Loss of Human Leukocyte Antigen Class II Expression in Immune-Privileged Site-Associated B-Cell Lymphoma

Authors' Affiliations: Departments of ¹ Pathology and Laboratory Medicine and ² Clinical Genetics, University Medical Center Groningen, Groningen, the Netherlands; ³ Department of Bioinformatics, Agendia B.V.; ⁴ Department of Pathology, Netherlands Cancer Institute, Amsterdam, the Netherlands; ⁵ Department of Pathology, Leiden University Medical Center, Leiden, the Netherlands; and ⁶ Department of Pathology, University of Wuerzburg, Wuerzburg, Germany

Requests for reprints: Marije Booman, Department of Pathology, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, the Netherlands. Phone: 31-50-3611284; Fax: 31-50-3632510; E-mail: m.booman{at}path.umcg.nl.

	Abstract

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Purpose and Experimental Design: Loss of human leukocyte antigen (HLA) expression on tumor cells is frequent in diffuse large B-cell lymphoma (DLBCL) arising in immune-privileged sites, such as the testis and central nervous system, and is associated with small homozygous deletions of HLA-DQ/HLA-DR and larger hemizygous deletions of the MHC region. To better understand the significance of down-regulation of HLA class II expression in relation to the homozygous and hemizygous deletions, we analyzed global gene expression patterns in a series of 26 testicular DLBCL after characterization of these deletions.

Results: Low levels of HLA-DR mRNA in whole testicular DLBCL samples were associated with a strong down-regulation of numerous immune-related genes specific for T cells, macrophages, antigen presentation and processing, lymphocyte activation, chemokines and chemokine receptors, and the complement system. The number of CD3⁺ tumor-infiltrating T cells was also significantly lower in low expressors of HLA-DR mRNA. Interestingly, hemizygous and homozygous deletions in the MHC region did not have any additional effect on global gene expression.

Conclusion: In conclusion, we found that loss of HLA class II mRNA expression in testicular DLBCL is associated with a significant change in global gene expression patterns. This effect is independent of the mechanism causing the down-regulation of HLA class II genes in the lymphoma cells.

We previously described the loss of HLA class I and II expression on tumor cells in >50% of the primary IP-DLBCL of the testis and central nervous system (3–6). In both IP-DLBCL subtypes, small homozygous deletions affected the HLA-DR and HLA-DQ genes, whereas larger hemizygous deletions affected the whole MHC region, including class I and III regions, as well. The specificity of this down-regulation in testicular DLBCL was confirmed in a recent immunohistochemical study on a large series of primary extranodal DLBCL presenting at various sites (7). In another recent study on predominantly primary nodal (non-IP) DLBCL, Rimsza et al. (8) showed that loss of expression of HLA class II is highly discriminative for the clinical behavior of the disease and by far the most significant single prognostic factor. Furthermore, loss of HLA-DR expression correlated with a low number of CD8⁺ T cells, suggesting that a presumed loss of tumor surveillance may have a negative effect on patient outcome.

To better understand the significance of down-regulation of HLA class II expression in relation to the homozygous and hemizygous deletions, we studied a series of primary testicular DLBCL by gene expression microarray analysis after detailed characterization of the hemizygous and homozygous deletions. Additionally, we studied the gene expression patterns in testicular DLBCL with low and high HLA-DR mRNA expression.

Here, we show that compared with normal B cells and primary nodal DLBCL, HLA-DR and HLA-DQ expression is dramatically down-regulated in most testicular lymphomas. Whereas neither homozygous nor hemizygous deletions have any major additional effect on overall gene expression signatures, down-regulation of HLA-DR is associated with a strong reduction of expression of many immune-associated genes and the number of infiltrating T cells.

	Materials and Methods

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Tissue sample collection and selection. Frozen and paraffin-embedded lymphoma samples from 26 primary testicular DLBCL and 10 primary nodal DLBCL according to the REAL and WHO classifications (9, 10) were collected from the tissue banks at the University Medical Center Groningen, Leiden University Medical Center, Josefine Nefkens Institute, Rotterdam, the Netherlands (courtesy Dr. L. Looijenga), and Netherlands Cancer Institute, Amsterdam, the Netherlands. All patients were immunocompetent, and patients with testicular DLBCL had stage IE or IIE disease. Seven of 26 testicular DLBCL (see Table 1 ) have been characterized previously by fluorescence in situ hybridization (FISH) and immunohistochemistry (3) and were included because of the presence of deletions. The median percentage of tumor cells present in frozen tissue sections as assessed on H&E-stained slides was 60%, with two testicular DLBCL cases having a tumor percentage of <50%.

Detection and quantification of T cells was done as described previously (7). In brief, sections were stained with polyclonal anti-CD3 (A0452, DAKO, Copenhagen, Denmark) and monoclonal anti-granzyme B (11F1, Sanbio, Uden, the Netherlands). The number of CD3-positive and granzyme B–positive lymphocytes was quantified using a video overlay–based measuring system (Q-PRODIT, Leica, Cambridge, United Kingdom) and expressed as the number of positive cells per 1 mm² of tumor.

Interphase FISH. Nuclei were isolated from frozen tissue sections and centrifuged onto glass slides. A centromeric 6 probe (D6Z1, Oncor, Gaithersburg, MD) was labeled with biotin-16-dUTP (Roche, Basel, Switzerland) by standard nick translation and cohybridized with locus-specific probes, which were labeled with digoxygenin-12 dUTP (Roche). These included PAC 93N13 (RCPI-1 library) for HLA-DQA and DRB (class II), cosmid M13A (American Tissue Culture Center) for tumor necrosis factor- (class III), and cosmids C109K2118 and C109B0233 (Imperial Cancer Research Fund chromosome 6 library) for HLA-A (class I). Hybridization and immunodetection were done as previously described (12).

In each DLBCL, signals were counted in 100 nuclei. As a control, each probe combination was hybridized on nuclei from nine routine hyperplastic tonsil samples. Cutoff levels for the detection of hemizygous or homozygous loss were determined as the average percentage of nuclei harboring a deletion in these nine tonsil samples plus thrice the SD. Cutoff levels for the presence of hemizygous deletions of MHC class I, III, and II probes were thus set at 27%, 36%, and 32% of nuclei harboring a deletion, respectively. Cutoff levels for homozygous deletions ranged from 0% to 6%.

Chromosome 6 array comparative genomic hybridization and data analysis. The chromosome 6 comparative genomic hybridization array contained 358 genomic clones mapping to chromosome 6, which were selected from the 1-Mb BAC collection obtained from Dr. N. Carter (Wellcome Trust Sanger Institute, United Kingdom; ref. 13), the Human BAC Resource Consortium_1 Set (Dr. P. de Jong, Children's Hospital Oakland Research Institute), and the RPCI-1 PAC library. In addition, the array contained 445 clones selected from a subtelomere array (14). Detailed information on the clone set of this array is available in Supplementary Table S1.

Genomic DNA was isolated from frozen tissue sections using the high salt method after overnight SDS/proteinase K digestion. Labeling and hybridization were done as previously described (14). DLBCL DNA was cohybridized with a standard reference consisting of a pool of either 20 normal male or 20 normal female genomic DNA samples. Data extraction, normalization, and analysis were done as described previously (14).

cDNA microarray hybridization. RNA isolation, linear amplification, and hybridization of 18K cDNA microarrays were done as described previously (15, 16). Detailed protocols can also be found at http://www.nki.nl/nkidep/pa/microarray/protocols.htm. Microarray slides containing >18,000 cDNA clones from the Research Genetics Human Sequence Verified library were prepared at the central microarray facility of the Netherlands Cancer Institute. Detailed information on the clone set can be found at http://microarray.nki.nl/download/geneid.html. These arrays were chosen for reasons of availability and their coverage of the MHC region. Twenty-six testicular and 10 nodal DLBCL samples were each cohybridized with a standard reference of pooled and amplified total RNA from five hyperplastic tonsils acquired by routine tonsillectomy. Each hybridization was repeated as a dye reversal. Ten percent of the experiments consisted of self-self hybridizations of the standard reference as a control for the experimental variation.

Fluorescent signals were extracted from the arrays by a DNA Microarray Scanner (Agilent, Palo Alto, CA) and quantified using Imagene 5.5 software (Biodiscovery, El Segundo, CA). The resulting data were normalized and corrected for various biases according to Yang et al. (17). Ratios of expression in DLBCL compared with tonsil were calculated, and weighed averages and confidence levels were calculated according to the Rosetta error model (18). Based on MA plots and the results from the error model, spots with an average intensity measurement A < 7 over all experiments were excluded from further analysis due to the fact that ratio measurements from those spots show too large a variation to be reliable.

Gene expression data analysis. Unsupervised clustering of genes and lymphoma samples was done using hierarchical clustering in Genesis (19). Using the Pearson correlation coefficient or Euclidian distance, complete similarity metrics were calculated based on expression levels in all tumors or in all genes for clustering of genes and experiments, respectively. Supervised clustering was done using significance analysis of microarrays (20). The median false discovery rate was used as a measure of significance. The results of supervised clustering were visualized using Genesis. Scatterplots of mean and median gene expression levels of different DLBCL groups were constructed in R (R Foundation for Statistical Computing, Vienna, Austria). Additional t statistics for selected genes were calculated using Welch's t test and visualized in scatterplots using Prism 3 (GraphPad Software, Inc., San Diego, CA). Functional relationships and signal transduction pathways, which involve selected genes, were analyzed in Pathway Assist version 3.0 (Iobion Labs, La Jolla, CA).

	Results

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HLA expression and deletions in the MHC region. Primary testicular and primary nodal DLBCL were characterized for HLA protein expression on the tumor cells and for genomic deletions of the MHC region by interphase FISH. Results are summarized in Table 1. Loss of expression of HLA-A/G and HLA-DR together with a high frequency of hemizygous and/or homozygous deletions in testicular DLBCL (3) was confirmed in this extended series.

To determine the boundaries of the hemizygous deletions that often extend beyond the MHC region more in detail, 11 testicular DLBCL for which genomic DNA was available were hybridized to a chromosome 6–specific comparative genomic hybridization array (Table 1). Figure 1 shows the clone density of the array for chromosome 6 and the deletions found in the MHC region in 7 of these 11 cases, including six cases with a proven deletion as determined by interphase FISH. All but one of these seven cases harbored an extensive hemizygous deletion of the MHC region. Additionally, in case 19T, a small homozygous deletion could be distinguished. Mapping of the hemizygous deletions showed a highly variable length towards the telomere 6p; in four of the seven cases, it did not extend further than 0.5 Mb telomeric of the MHC class I region. Most interestingly, at the centromeric side, all larger deletions ended in a region of 1 Mb between two BAC clones (RP3-468B3 and RP3-349A12) located 0.5 and 1.4 Mb centromeric of the MHC class II region.

View larger version (17K): [in this window] [in a new window]   Fig. 1. Comparative genomic hybridization array results for chromosome 6p. Top, clone density is indicated beneath the base position. Narrow bar, hemizygous deletions; broad bar, homozygous deletions. Shaded box, MHC region. Bottom, close-up of the MHC region. Stars, locations of probes used in FISH analysis.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Supervised cluster analysis of testicular and nodal DLBCL. Top, supervised cluster analysis of testicular (orange) and nodal (blue) DLBCL resulted in 431 clones that are differentially expressed. Right margin, cluster containing HLA class II genes. Presence of deletions and HLA-DR protein expression (top) for each case. FISH results: +, deletion present; ±, deletion of borderline significance (see Table 1); –, no deletion; extended hemi, hemizygous deletion of MHC class I, II, and II regions; class II homo, homozygous deletion of HLA class II. HLA-DR protein expression (in tumor cells, determined by immunohistochemistry): +, protein present; –, protein absent. Bottom, HLA class II cluster enlarged. Right margin, individual HLA class II clones.

6p deletions have a limited effect on global gene expression profiles of testicular DLBCL. We analyzed effects of the hemizygous 6p deletions involving the MHC region on global gene expression in testicular DLBCL. To that end, we selected five cases without any deletions (3T, 9T, 12T, 15T, and 21T) and six cases with large hemizygous deletions in a high percentage of nuclei (1T, 4T, 5T, 7T, 8T, and 10T) as determined by interphase FISH. There was no further selection based on HLA protein or mRNA expression. Within the latter group, all cases had deletions in the MHC class II and III regions, and four of six had additional deletions of the class I region (Table 1). Although no significant differences between these two groups were found in a supervised clustering analysis using significance analysis of microarray, it is noteworthy that 5 of the 11 low expressed genes selected at a minimum false discovery rate of 33% are located at chromosome 6p, including three at 6p21.3: DEK (6p22.3), GNL1, TUBB, DOM3Z (6p21.3), and SLC35B2 (6p21.1).

We separately analyzed the possible effects of the small homozygous deletions on gene expression of the involved HLA genes and possible downstream targets. Five testicular DLBCL with a homozygous deletion in a high percentage of nuclei (2T, 4T, 6T, 8T, and 19T) were chosen to compare to five cases without any deletions (3T, 9T, 12T, 15T, and 21T), again with no further selection for HLA protein or mRNA expression level. The mean gene expression values for each group were visualized in a scatterplot (Fig. 3 ), illustrating that the two groups are highly similar. No significant differences in gene expression between both groups were found with significance analysis of microarray (data not shown). Expression of classic HLA class II genes in both the deletion-positive and deletion-negative testicular DLBCL groups was three to four times lower than in the reference tonsil sample, confirming down-regulation of these genes irrespective of deletions. There is a trend for the HLA-DQ and HLA-DR genes towards an even lower expression in cases with a homozygous deletion. Expression levels of non-classic HLA genes were equal to that in the reference tonsil sample.

View larger version (23K): [in this window] [in a new window]   Fig. 3. HLA class II gene expression in testicular DLBCL with and without homozygous deletions. For each gene, the average log ratio in testicular DLBCL with homozygous deletions of HLA-DQ/DR genes (2T, 4T, 6T, 8T, and 19T) is plotted against the average log ratio in testicular DLBCL without deletions (10T, 7T, 1T, 8T, 5T, and 4T). Solid lines, equal expression level; dotted lines, 2-fold difference. Big black dots, non-classic HLA class II genes; black star, classic HLA class II genes.

Correlation between HLA-DR mRNA levels and immune signatures. Subsequently, we investigated the relation between HLA-DR mRNA expression and immune-mediated responses as measured by other gene expression studies in nodal DLBCL. Monti et al. (21) have distinguished three types of nodal DLBCL, of which one is characterized by a high expression of so-called "host response" genes and a large number of infiltrating inflammatory and immune cells. The Staudt group has defined a "lymph node signature" in nodal DLBCL, of which a selection of genes was associated with good prognosis (22, 23). We compared these two signatures with our own set of genes that are down-regulated in low expressors of HLA-DR mRNA. Fifty-two of 72 genes from the "host response signature" were present on our array, of which 24 were down-regulated in testicular DLBCL with low HLA-DR mRNA expression. One hundred sixteen of 141 genes from the "lymph node signature" were present on the array of which 31 were down-regulated. The genes with a direct immunologic function are indicated in Table 2. Only three genes tended towards a higher expression in cases with low HLA-DR mRNA expression: CD14 (Table 3A), cystatin C, and granulysin. Finally, the 116 genes from the "lymph node signature" were able to discern testicular DLBCL with high or low levels of HLA-DR expression in a hierarchical cluster analysis (data not shown).

To investigate if the number of infiltrating T cells is related to the apparent down-regulation of the immune response, we compared numbers of CD3-positive T cells and granzyme B-positive cells between four testicular DLBCL with high HLA-DR mRNA expression (cases 5T, 16T, 17T, and 20T) and four with low HLA-DR mRNA expression (cases 1T, 2T, 4T, and 8T). These cases were included in the study by Riemersma et al. (7) using Welch's t test. Cases with low expression contained a significantly lower number of T cells, but there was no difference in the total number of granzyme B–positive activated cytotoxic T cells and natural killer cells (Table 3B).

	Discussion

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Recent gene expression studies have suggested that down-regulation of HLA class II expression has a major biological effect reflected in clinical tumor characteristics and outcome. This has been shown for B-cell lymphomas, that naturally express HLA class II on lymphoma cells (8, 22), but also for carcinomas in which HLA class II can be expressed on the antigen-presenting cells within the tumor (24). It is increasingly appreciated that not only HLA class I but also class II is essential for mounting an adequate antitumor immune response. Antigen presentation via HLA class II is indispensable to activate a CD4⁺ T-cell population that may induce a CD8⁺ T cell–mediated cytotoxic antitumor response (25, 26) and recruit additional effector cell populations, such as macrophages (26, 27). In carcinomas, an immune-mediated antitumor response is mainly mediated by professional antigen-presenting cells.

In B-cell lymphomas, both professional antigen-presenting cells as well as the tumor B cells may play an active role in the antitumor response by cytotoxic T cells, whereas the tumor cells might also be dependent on T helper cells and/or tumor promoting factors produced by macrophages. This generates a complex and intimate immunologic relationship between tumor cells and surrounding reactive cells. The significance of this immunologic context of DLBCL has recently been underlined by gene expression studies with the identification of a "host response signature" and "lymph node signature" in these lymphomas (21, 22). Although these phenomena might be tightly linked to the expression of HLA class II on the tumor cells and/or antigen-presenting cells, this has not been studied extensively. Our data show that the loss of expression of HLA-DR at the mRNA level in whole testicular DLBCL samples is associated with a significantly lower expression of many immune-related genes. Detailed analysis revealed numerous genes involved in different aspects of the immune response, including markers for both T cells, natural killer cells, macrophages, and antigen-presenting cells (Table 2). The coordinate down-regulation of these genes with HLA-DR levels indicates a severe disruption of the immune response in testicular DLBCL with low HLA-DR mRNA expression levels. Fifty percent of the HLA-DR coregulated genes with an immune function in our study overlap with the "host response" and "lymph node" signatures published by other groups (21, 22), supporting the relevance of this finding.

To confirm our data on a different platform, we independently analyzed four of the testicular DLBCL with the lowest and five of the cases with the highest HLA-DR mRNA expression levels on the Affymetrix U133 2.0 Plus array. We again found a down-regulation of genes involved in immune recognition in the low expressors. Sixty-eight of the 286 differentially expressed genes (24%) had an immune-related function, of which 32 were already identified in cDNA array analysis.⁷

Intriguingly, in studies published by Shipp et al. (21, 28), HLA class II gene expression levels were neither associated with clinical outcome nor with their "host response profile." This could possibly be attributed to technical dissimilarities: whereas we and the LLMPP group used cDNA arrays that gave consistent results for multiple clones of the same HLA class II genes, Monti et al. (21) used Affymetrix oligo arrays, and in the supplementary data set published with their article, different probe sets for the same HLA class II genes were not consistently assigned to one expression signature. This might be due to the fact that most Affymetrix probe sets for HLA class II genes, indicated by _x_at, "may cross-hybridize in an unpredictable manner" (ref. 29, p. 94).

Using immunohistochemistry on a subset of our cases, a low level of HLA-DR mRNA expression was associated with a significantly lower number of infiltrating CD3⁺ cells and a slightly lower number of granzyme B–positive cells (Table 3B). This is in concordance with the low levels of TCR and TCRß mRNA in these cases as assessed by microarray analysis (Table 3A) and indicates that the low expression of immune response–associated genes can partly be attributed to a lower number of infiltrating T cells. In a recent investigation on 254 DLBCL, including 18 HLA-DR protein positive and 56 negative testicular DLBCL, no significant correlation between HLA-DR protein expression on the tumor cells and the numbers of infiltrating T cells was found (7). These seemingly contradictory data suggest that a concerted down-regulation of HLA-DR on both the tumor cells and surrounding antigen-presenting cells, and not HLA expression on only the tumor cells, is essential in debilitating the immune response. Because we could study gene expression in only two cases with retained HLA class II protein expression on the tumor cells, we could not further explore this issue on the gene expression level.

Several macrophage- and monocyte-related genes, including CD68, were down-regulated in testicular DLBCL with low HLA class II mRNA levels. Intriguingly, CD14 showed up-regulation in this group (Table 3A). In Burkitt's lymphoma, CD14 is known to be involved in IL-10-induced phagocytosis of apoptotic material and has an anti-inflammatory effect. Moreover, CD14-positive macrophages produce BAFF to inhibit apoptosis of tumor cells (30). The properties of these tumor-infiltrating macrophages are reminiscent of polarized M2 macrophages (31). We hypothesize that increased CD14 levels in macrophages in testicular DLBCL with low HLA-DR mRNA expression might promote lymphoma growth and survival and thus might be related to the poor prognosis associated with HLA class II–negative DLBCL. This possible tumor survival mechanism remains to be elucidated in testicular DLBCL.

In many IP-DLBCL arising at immune-privileged sites, such as the testis and central nervous system, but also in a minority of nodal (non-IP) DLBCL, down-regulation of HLA class II is effected by hemizygous and homozygous deletions and/or mitotic recombination at chromosome 6p21.3 (3, 4). Using interphase and DNA fiber FISH, we previously showed that the small homozygous deletions only involve the HLA-DQ and HLA-DR genes (3). Gene expression analysis now indicates that expression of HLA class II mRNA is very low in testicular DLBCL in general, and that the small homozygous deletions result only in a slightly further down-regulation. The mechanism by which HLA class II down-regulation is accomplished in testicular DLBCL without homozygous deletions remains to be elucidated. Very recently Rimsza et al. reported that in non-IP DLBCL loss of HLA class II mRNA expression is not due to chromosomal deletions but rather correlates with loss of expression of MHC class II transactivator and invariant chain genes, indicating loss of transcriptional activation (32). We explored this for our testicular DLBCL without homozygous deletions. Indeed, there was a significant correlation between expression of MHC class II transactivator and HLA class II genes in all 10 testicular DLBCL without a homozygous deletion (r ranges from 0.65 for HLA-DQ to 0.72 for HLA-DR using the Spearman correlation), whereas this correlation was absent in the 13 cases with a significant homozygous deletion. These results confirm that multiple mechanisms are involved in the down-regulation of HLA class II genes in DLBCL. The intriguing question why so many IP-DLBCL use a deletion instead of transcriptional down-regulation remains to be answered. Perhaps the tumor cells of IP-DLBCL are more dependent on a shutdown of the immune response and therefore need a more permanent loss of HLA class II expression as mediated by physical deletion of the HLA class II–encoding genes.

Hemizygous deletions are much larger than the homozygous deletions and may target many other genes than the classic HLA genes at chromosome 6p. Comparative genomic hybridization array analysis showed that the minimal region of interest on chromosome 6p is restricted to a region of 5.0 Mb starting 0.5 Mb telomeric of MHC class I and extending 1 Mb centromeric of MHC class II. Global gene expression patterns showed a very limited effect of the hemizygous deletions, suggesting that no specific genes in this region are targeted.

Previous studies on predominantly nodal DLBCL have shown that loss of expression of HLA class II is an independent adverse prognostic factor (8). The present series of testicular DLBCL is not suitable for a meaningful correlative study on the relationship between HLA class II expression and clinical behavior because the cases were collected over a very long period of time and treatment was highly variable.

In conclusion, we found that loss of HLA class II mRNA expression in lymphoma cells and the surrounding infiltrate is associated with a significant change in global gene expression patterns reflecting a decrease in the immune response, which is paralleled by a decrease in the relative number of infiltrating reactive T cells. This effect is independent of the mechanism causing the down-regulation of HLA-DR on the lymphoma cells.

	Acknowledgments

 
We thank L. Delahaye, A. Witteveen, P. van der Vlies, and T. Dijkhuizen for their help with hybridizations of the cDNA and comparative genomic hybridization arrays.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

⁷ M. Booman and A. Rosenwald, unpublished results.

Received 11/29/05; revised 2/ 3/06; accepted 3/ 2/06.

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