Locus specific DNA methylation in human immunological responses

All cells in the human body contains the same genome. Yet, there are hundreds of different cell types, with widely different phenotype, and function. The differential gene expression leading to this diversity is tightly regulated by epigenetics, i.e. modifications to the genome without interfering with the sequence. There are a number of epigenetic modifications. In this thesis, I have studied DNA methylation as a readout for lineage specificity or effector function in cells from the immune system, in clinical settings. In paper I we develop a method based on DNA methylation analysis to determine the lineage commitment of the CD4+ T helper cells. First, we establish a regulatory site in the promotor of the IL17A gene. Next, we use signature genes IFNG, IL13, IL17A and FOXP3, to determine lineage commitment towards corresponding T helper cell subsets Th1, Th2, Th17 and Tregs. We call this method EILA (Epigenetic immune lineage assay), and demonstrate that it is usable in clinical samples from rheumatoid arthritis and multiple sclerosis. In paper II, we take advantage of the same CD4+ T cell specific loci to investigate the adaptive immune response in and around the tumour microenvironment in specimens derived from patients with urinary bladder cancer (UBC). By sorting and examining CD4+ T cells from blood, tumour and regional lymph nodes, we conclude that patients with higher degree of lineage commitment have a better prognosis. Furthermore, we demonstrate that patients responding to neoadjuvant chemotherapy have a larger proportion of commitment cells, post treatment, compared with the non-responders. In paper III we further examine the same patient material from UBC patients, but instead focus on the cytotoxic features of CD8+ T lymphocytes. We establish a methylation pattern in the perforin gene PRF1 predictive for protein expression and deploy this locus as a readout for cytotoxic functionality. We demonstrate that the tumour infiltrating CD8+ T cells are pre- dispositioned to be cytotoxic through PRF1 demethylation, but that they lack corresponding protein expression, and show signs of exhaustion. The cells demonstrating a TRM phenotype, can still be woken anew, upon in vitro re-stimulation, demonstrating that they are not terminally exhausted. In paper IV we investigate whole blood leucocytes and the DNA methylation status of the glucocorticoid gene NR3C1. In contrast to literature studies on healthy volunteers, our cohort of surgical patients demonstrate a homogeneous pattern of demethylation in the previously described CpG island of NR3C1. As opposed to our hypothesis, we found no correlation between methylation and clinical outcome post-surgery in this patient cohort. Nevertheless, when employing multifactorial analysis to investigating the impact of genotype we found four single nucleotide polymorphisms that influenced the outcome. In conclusion, this thesis demonstrate multiple ways in which DNA methylation analysis can be used to read the immune system, but also that the loci selected for investigation has to be carefully chosen following thorough functional investigations. The results presented herein can contribute to further development of treatments to a variety of clinical conditions

Urothelial bladder cancer may suppress perforin expression in CD8+ T cells by an ICAM-1/TGFβ2 mediated pathway

The immune system plays a significant role in urothelial bladder cancer (UBC) progression, with CD8+ T cells being capable to directly kill tumor cells using perforin and granzymes. However, tumors avoid immune recognition by escape mechanisms. In this study, we aim to demonstrate tumor immune escape mechanisms that suppress CD8+ T cells cytotoxicity. 42 patients diagnosed with UBC were recruited. CD8+ T cells from peripheral blood (PB), sentinel nodes (SN), and tumor were analyzed in steady state and in vitro-stimulated conditions by flow cytometry, RT-qPCR, and ELISA. Mass spectrometry (MS) was used for identification of proteins from UBC cell line culture supernatants. Perforin was surprisingly found to be low in CD8+ T cells from SN, marked by 1.8-fold decrease of PRF1 expression, with maintained expression of granzyme B. The majority of perforin-deficient CD8+ T cells are effector memory T (TEM) cells with exhausted Tc2 cell phenotype, judged by the presence of PD-1 and GATA-3. Consequently, perforin-deficient CD8+ T cells from SN are low in T-bet expression. Supernatant from muscle invasive UBC induces perforin deficiency, a mechanism identified by MS where ICAM-1 and TGFβ2 signaling were causatively validated to decrease perforin expression in vitro. Thus, we demonstrate a novel tumor escape suppressing perforin expression in CD8+ T cells mediated by ICAM-1 and TGFβ2, which can be targeted in combination for cancer immunotherapy

Tc1 conditions can restore perforin expression in CD8⁺ T cells from sentinel nodes.

<p>CD8⁺ T cells sorted from sentinel node (SN) were cultured in Tc1 conditions <i>in vitro</i> for seven days in order to rescue perforin expression. These SN-derived CD8⁺ T cells were stimulated with anti-CD3 and anti-CD28 stimulating antibodies with the presence of IL-12 and IL-2 cytokines, as well as an anti-IL-4 neutralizing antibody. At the end of the culture, cells were analyzed by flow cytometry and RT-qPCR. (<b>A</b>) Dot plots showed the flow cytometry data from a representative patient for granzyme B vs. perforin expression, before and after the stimulation. The gate was based on isotype control and the frequency of granzyme B and perforin expression was counted out of CD8⁺ T cells. (<b>B</b>) Flow cytometry result of T-bet expression percentage from CD8⁺ T cells pre- and post-stimulation was analyzed. The frequency of T-bet expression was calculated from CD8⁺ T cells. (<b>C</b>) <i>TBX21</i> and <i>GATA3</i> gene expression analysis was done by RT-qPCR from cells in different culture conditions. <i>RPII</i> gene was used as housekeeping gene and the fold change was calculated based on cells without IL-12 and anti-IL-4 as control using 2^-ΔΔCt method.</p

Production and secretion of perforin in SN CD8⁺ T cells are low after <i>in vitro</i> reactivation.

<p>Lymphocytes isolated from peripheral blood (PBMC) and sentinel node (SN) were cultured for seven days with addition of autologous tumor homogenate. (<b>A</b>) Flow cytometry was done to phenotype the co-expression in CD8⁺ T cells from PBMC and SN before and after reactivation. The results were shown in dot plots and gated based on isotype control. The frequency of granzyme B and perforin expression was counted out of CD8⁺ T cells. Dot plots showed data from a representative cystectomized patient. (<b>B</b>) Intracellular perforin was measured by Median Fluorescence Intensity (MFI) post 7-day culture using flow cytometry from (A). The data are means with error bars indicating SEM. Mann-Whitney was used as the statistical test. (<b>C</b>) The concentrations (pg/ml) of secreted granzyme B and perforin after seven days of culture were analyzed by ELISA and compared between <i>in vitro</i> culture supernatants of PBMC and SN. The data are means with error bars indicating SEM. Mann-Whitney was used as the statistical test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p

ICAM-1 and TGFβ2 signal from muscle invasive UBC causes perforin downregulation.

<p>Culture supernatants of urothelial bladder cancer (UBC) cell lines were acquired from RT4 (non-muscle invasive) and 5637 (muscle invasive) cell lines. CD8⁺ T cells were then isolated from peripheral blood of healthy donor and cultured in <i>vitro</i> with these supernatants for five days. (<b>A</b>) Analysis of perforin coding gene (<i>PRF1</i>) expression was done by RT-qPCR. mRNA was extracted post-culture from the cells of the culture groups. Bar graphs show different expression of <i>PRF1</i> in CD8⁺ T cells cultured <i>in vitro</i> between RT4 (non-muscle invasive) and 5637 (muscle invasive) supernatant. <i>RPII</i> gene was used as housekeeping gene and the fold change was calculated in regards of RT4 medium using 2^-ΔΔCt method. The data are means with error bars indicating SEM. Paired-t-test was used as the statistical test. (<b>B</b>) Flow cytometry analysis of CD8⁺ T cells at the end of culture was done. The results comparing three groups were shown in dot plots from a representative healthy donor and gated based on isotype control. (<b>C</b>) The frequency of perforin^- CD8⁺ T cells from (B) was counted out of CD8⁺ T cells. The data are means with the error bars indicating SEM. One-way repeated-measure ANOVA was used as the statistical test. (<b>D</b>) Mass spectometry (MS) analysis identified proteins expressed by RT4 and 5637 cell line. Proteins under the category “immune system process” on the GO (Gene Ontology) term were selected for network analysis based on STRING database. Size represented differential expression between RT4 and 5637 supernatants and the color represented betweenness which marked the influence of the protein to the network. Color indicators: blue = low, yellow = average and red = high. (<b>E</b>) The expression of ICAM-1 was validated by flow cytometry on RT4 and 5637 cell line. RT4 and 5637 cells were identified by EpCAM expression. (<b>F</b>) Validation of perforin downregulation by ICAM-1 and TGFβ2 was done <i>in vitro</i> on CD8⁺ T cells isolated from healthy donors in the presence of anti-CD3 stimulating antibody for 5 days. Perforin coding gene (<i>PRF1</i>) expression was done by RT-qPCR. mRNA was extracted post-culture from the cells. Bar graphs show different expression of <i>PRF1</i> in CD8⁺ T cells cultured <i>in vitro</i> between control and TGFβ2 + ICAM-1 + αCD3. RPII gene was used as housekeeping gene and the fold change was calculated in regards of blank medium using 2-ΔΔCt method. The data are means with error bars indicating SEM. Paired-t-test was used as the statistical test. (<b>G</b>) Flow cytometry analysis of CD8⁺ T cells at the end of culture was done. The results were shown in dot plots and gated based on isotype control from a representative healthy donor. The frequency of granzyme B and perforin expression was counted out of CD8⁺ T cells. (<b>H</b>) The frequency of granzyme B⁺/perforin^- expressing cells from (G) was counted out of CD8⁺ T cells. The data are means with error bars indicating SEM. Paired-t-test was used as the statistical test. * p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p

Sentinel node CD8⁺ T cells with perforin deficiency are exhausted Tc2 cells.

<p>(<b>A</b>) CD8⁺ T cells isolated from sentinel node (SN) were further phenotyped using flow cytometry to demonstrate the difference in T cells exhaustion markers expression (PD-1) and Tc1 transcription factor (T-bet) between granzyme B⁺/perforin⁻CD8⁺ T cells (green box) and granzyme B⁺/perforin⁺ CD8⁺ T cells (red box). The expression of PD-1 and T-bet were shown in dot plots from a representative patient and gated based on isotype control. (<b>B</b>) The frequency of PD-1 and T-bet from (A) was calculated either out of granzyme B⁺/perforin⁻or granzyme B⁺/perforin⁺ CD8⁺ T cells. The data are means with the error bars indicating SEM. Mann-Whitney was used as the statistical test. (<b>C</b>) The expression of T-bet, encoded by <i>TBX21</i> gene, was compared among CD8⁺ T cells sorted from peripheral blood mononuclear cells (PBMC), sentinel node (SN), and tumor. mRNA was extracted from the sorted cells and the <i>TBX21</i> gene expression was analyzed by RT-qPCR. The expression of <i>TBX21</i> was quantified using 2^-ΔΔCt method and the fold change was calculated in regards of PBMC as control. <i>RPII</i> gene was used as housekeeping gene. The data are means with error bars indicating SEM. Kruskal-Wallis was used as the statistical test. (<b>D</b>) Same as in (C), but the analysis was done on <i>GATA3</i> gene expression. (<b>E</b>) The frequency of naïve T cells (CD45RA⁺ CCR7⁺), central memory T (T_CM) cells (CD45RA^- CCR7⁺), effector memory T (T_EM) cells (CD45RA^- CCR7^-), and effector memory T with CD45RA expression (T_EMRA) cells (CD45RA⁺ CCR7^-) was calculated either out of granzyme B⁺/perforin⁻or granzyme B⁺/perforin⁺ CD8⁺ T cells. The data are means with the error bars indicating SEM. Kruskal-Wallis was used as the statistical test. * p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p

Perforin deficiency in CD8⁺ T cells from sentinel nodes.

<p>(<b>A</b>) The expression of granzyme B and perforin in CD8⁺ T cells of different tissues were phenotyped by flow cytometry. The co-expression pattern in CD8⁺ T cells was shown in dot plots and gated for distinguishing between double and single expression of granzyme B and perforin. The gate was based on isotype control and the frequency of granzyme B and perforin expression was counted out of CD8⁺ T cells. Dot plots showed a representative data from a patient underwent transurethral resection of the bladder (TUR-B) and cystectomy. (<b>B</b>) The frequency of granzyme B⁺/perforin⁺ CD8⁺ T cells from PBMC, SN, and tumor tissues was shown in graphs and was calculated out of CD8⁺ T cells (n = 27). (<b>C</b>) Same as in (B) but the analysis was done on granzyme B⁺/perforin^- CD8⁺ T cells. The data are means with the error bars indicating SEM. Kruskal-Wallis was used as the statistical test. (<b>D</b>) The expression of gene responsible in encoding granzyme B (<i>GZMB</i>) and perforin (<i>PRF1</i>) in CD8⁺ T cells isolated from PBMC, SN, and tumor (n = 6). RT-qPCR was done to analyze the gene expression followed by quantification using 2^-ΔΔCt method. The fold change was calculated in regards of PBMC as control, with <i>RPII</i> gene used as the housekeeping gene. The data are the means of Log₂ of fold change (2^-ΔΔCt) with the error bars indicating SEM. Kruskal-Wallis was used as the statistical test on each gene. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p

Increased CD4+ T cell lineage commitment determined by CpG methylation correlates with better prognosis in urinary bladder cancer patients

BACKGROUND: Urinary bladder cancer is a common malignancy worldwide. Environmental factors and chronic inflammation are correlated with the disease risk. Diagnosis is performed by transurethral resection of the bladder, and patients with muscle invasive disease preferably proceed to radical cystectomy, with or without neoadjuvant chemotherapy. The anti-tumour immune responses, known to be initiated in the tumour and draining lymph nodes, may play a major role in future treatment strategies. Thus, increasing the knowledge of tumour-associated immunological processes is important. Activated CD4+ T cells differentiate into four main separate lineages: Th1, Th2, Th17 and Treg, and they are recognized by their effector molecules IFN-γ, IL-13, IL-17A, and the transcription factor Foxp3, respectively. We have previously demonstrated signature CpG sites predictive for lineage commitment of these four major CD4+ T cell lineages. Here, we investigate the lineage commitment specifically in tumour, lymph nodes and blood and relate them to the disease stage and response to neoadjuvant chemotherapy. RESULTS: Blood, tumour and regional lymph nodes were obtained from patients at time of transurethral resection of the bladder and at radical cystectomy. Tumour-infiltrating CD4+ lymphocytes were significantly hypomethylated in all four investigated lineage loci compared to CD4+ lymphocytes in lymph nodes and blood (lymph nodes vs tumour-infiltrating lymphocytes: IFNG -4229 bp p &lt; 0.0001, IL13 -11 bp p &lt; 0.05, IL17A -122 bp p &lt; 0.01 and FOXP3 -77 bp p &gt; 0.05). Examination of individual lymph nodes displayed different methylation signatures, suggesting possible correlation with future survival. More advanced post-cystectomy tumour stages correlated significantly with increased methylation at the IFNG -4229 bp locus. Patients with complete response to neoadjuvant chemotherapy displayed significant hypomethylation in CD4+ T cells for all four investigated loci, most prominently in IFNG p &lt; 0.0001. Neoadjuvant chemotherapy seemed to result in a relocation of Th1-committed CD4+ T cells from blood, presumably to the tumour, indicated by shifts in the methylation patterns, whereas no such shifts were seen for lineages corresponding to IL13, IL17A and FOXP3. CONCLUSION: Increased lineage commitment in CD4+ T cells, as determined by demethylation in predictive CpG sites, is associated with lower post-cystectomy tumour stage, complete response to neoadjuvant chemotherapy and overall better outcome, suggesting epigenetic profiling of CD4+ T cell lineages as a useful readout for clinical staging

Additional file 3: of Increased CD4+ T cell lineage commitment determined by CpG methylation correlates with better prognosis in urinary bladder cancer patients

Figure S1. Analysis of Cisplatin effect on healthy donors CD4+ T cells in vitro. CD4+ T cells were isolated from blood of healthy donors (n = 4) and cultured in vitro in the presence of neoadjuvant chemotherapy drug, Cisplatin. Stimulation at day 0 is indicated on x-axis. Sim = αCD3 and αCD28. Cisp 25 μM cisplatin. At day 6, all cultures were treated with αCD3 and αCD28, and cisplatin cultures (grey bars) received 25 μM cisplatin. The cells were harvested at day 12 for analysis. a Whole genome methylation was measured by 5mC ELISA. Corresponding cultures without cisplatin was used for normalization. Friedman test was used for statistical analysis. b Methylation of IFNG locus was measured. Unstimulated cells from Day 0 was used as normalization. (TIF 840 kb