Per Marits

FOXP3 Promoter Demethylation Reveals the Committed Treg Population in Humans

Background Naturally occurring thymus derived regulatory T cells (Tregs) are central in the maintenance of self-tolerance. The transcription factor FOXP3 is crucial for the suppressive activity of Tregs and is considered the most specific marker for this population. However, human non regulatory T cells upregulate FOXP3 transiently upon activation which calls for other means to identify the Treg population. Since epigenetic mechanisms are involved in the establishment of stable gene expression patterns during cell differentiation, we hypothesized that the methylation profile of the FOXP3 promoter would allow the distinction of truly committed Tregs. Methodology/Principal Findings Human CD4+CD25hi Tregs displayed a demethylated FOXP3 promoter (1.4%±0.95% SEM methylated) in contrast to CD4+CD25lo T cells which were partially methylated (27.9%±7.1%). Furthermore, stimulated CD4+CD25lo T cells transiently expressed FOXP3 but remained partially methylated, suggesting promoter methylation as a mechanism for regulation of stable FOXP3 expression and Treg commitment. In addition, transient FOXP3 expressing cells exhibited suppressive abilities that correlate to the methylation status of the FOXP3 promoter. As an alternative to bisulphite sequencing, we present a restriction enzyme based screening method for the identification of committed Tregs and apply this method to evaluate the effect of various culturing conditions. We show that a partial demethylation occurs in long-term cultures after activation, whereas the addition of TGF-β and/or IL-10 does not induce any additional change in methylation level. Conclusions/Significance The unique FOXP3 promoter methylation profile in Tregs suggests that a demethylated pattern is a prerequisite for stable FOXP3 expression and suppressive phenotype. Presently, FOXP3 is used to identify Tregs in several human diseases and there are future implications for adoptive Treg transfer in immunotherapy. In these settings there is a need to distinguish true Tregs from transiently FOXP3+ activated T cells. The screening method we present allows this distinction and enables the identification of cells suitable for in vitro expansions and clinical use.

Profiling of CD4+ T Cells with Epigenetic Immune Lineage Analysis

Proper transcriptional control of pro- and anti-inflammatory responses of the immune system is important for a fine-tuned balance between protection and tolerance. Emerging evidence suggests a key role for epigenetic regulation in governing the Th cell differentiation, where effector cytokines direct the overall immune response. In this study, we describe a method to pinpoint the location of isolated human CD4+ T cells on any T cell effector axis based on specific CpG methylation of cytokine and transcription factor loci. We apply the method on CD4+ cells obtained from rheumatoid arthritis and multiple sclerosis patients and show that synovial fluid infiltrating CD4+ T cells are committed toward both Th1 and regulatory T cell phenotype, whereas the Th2 response is suppressed. Furthermore, we show that the IL-17A gene is regulated by promoter methylation and that Th17 commitment is not a common feature in the inflamed joints of rheumatoid arthritis patients. We conclude that the method described in this paper allows for accurate profiling of Th lineage commitment in ex vivo-isolated CD4+ T cells.

Increased antigen presenting cell-mediated T cell activation in mice and patients without the autoimmune regulator

Patients with autoimmune polyendocrine syndrome type I (APS I)suffer from endocrine and non‐endocrine disorders due to mutations in the autoimmune regulator gene (AIRE). Mouse Aire is expressed both in thymic medullary epithelial cells and in peripheral antigen‐presenting cells, suggesting a role in both central and peripheral tolerance. We here report that Aire–/– dendritic cells (DC) activate naive T cells more efficiently than do Aire+/+ DC. Expression array analyses of Aire–/– DC revealed differential regulation of 68 transcripts, among which, the vascular cell adhesion molecule‐1 (VCAM‐1) transcript was up‐regulated in Aire–/– DC. Concurrently, the expression of the VCAM‐1 protein was up‐regulated on both Aire–/– DC and monocytes from APS I patients. Blocking the interaction of VCAM‐1 prevented enhanced Aire–/– DC stimulation of T cell hybridomas. We determined an increased number of DC in spleen and lymph nodes and of monocytes in the blood from Aire–/– mice, and an increased number of blood monocytes in APS I patients. Our findings imply a role for Aire in peripheral DC regulation of T cell activation, and suggest that Aire participates in peripheral tolerance.

FOXP3 and survival in urinary bladder cancer

Whats known on the subject? and What does the study add?

CpG Methylation of the IFNG Gene as a Mechanism to Induce Immunosupression in Tumor-Infiltrating Lymphocytes

The execution of appropriate gene expression patterns during immune responses is of eminent importance where CpG methylation has emerged as an essential mechanism for gene silencing. We have charted the methylation status of regulatory elements in the human IFNG gene encoding the signature cytokine of the Th1 response. Surprisingly, human naive CD4+ T lymphocytes displayed hypermethylation at the IFNG promoter region, which is in sharp contrast to the completely demethylated status of this region in mice. Th1 differentiation induced demethylation of the IFNG promoter and the upstream conserved nucleotide sequence 1 enhancer region, whereas Th2-differentiated lymphocytes remained hypermethylated. Furthermore, CD19+ B lymphocytes displayed hypomethylation at the IFNG promoter region with a similar pattern to Th1 effector cells. When investigating the methylation status among tumor-infiltrating CD4+ T lymphocytes from patients with colon cancer, we found that tumor-infiltrating lymphocytes cells are inappropriately hypermethylated, and thus not confined to the Th1 lineage. In contrast, CD4+ T cells from the tumor draining lymph node were significantly more demethylated than tumor-infiltrating lymphocytes. We conclude that there are obvious interspecies differences in the methylation status of the IFNG gene in naive CD4+ T lymphocytes, where Th1 commitment in human lymphocytes involves demethylation before IFNG expression. Finally, investigations of tumor-infiltrating lymphocytes and CD4+ cells from tumor draining lymph node demonstrate methylation of regulatory regions within key effector genes as an epigenetic mechanism of tumor-induced immunosupression.

Trough s-infliximab and antibodies towards infliximab in a cohort of 79 IBD patients with maintenance infliximab treatment

BACKGROUND AND AIMS The anti-TNF antibody infliximab is effective in inducing remission in Crohns disease as well as in ulcerative colitis and many patients are treated for several years with sustained clinical remission. However, the role of monitoring s-infliximab and antibodies towards infliximab during maintenance treatment remains unclear. Our aim was to correlate serum drug levels and antibodies to clinical activity, CRP, albumin and concomitant immunosuppression in a cohort on maintenance infliximab treatment. METHODS We included 79 patients with Crohns disease or ulcerative colitis who had responded to infliximab and received maintenance treatment (4-69 infusions) in this retrospective study. Infliximab levels and antibodies towards the drug were analyzed with in-house-developed ELISA assays. RESULTS The mean s-infliximab was significantly higher in patients in remission (4.1μg/mL) as compared with disease flare (mean 1.8μg/mL); p<0.001. The s-infliximab showed a significant negative correlation with Harvey-Bradshaw index (r=-0.21; p<0.05). Serum-infliximab progressively decreased with the number of accumulated infusions (p<0.05). In patients with undetectable trough levels, 55% of the patients with concomitant immunosuppressive were positive for antibodies against infliximab, as compared with 94% of patients on monotherapy. Patients with undetectable serum-infliximab were in clinical remission at 25% of the visits. CONCLUSIONS The trough level 4.1μg/mL may serve as cut-off for clinical remission. Drug trough levels decreased during treatment and almost all patients with undetectable s-infliximab and monotherapy had developed antibodies against the drug.

Sentinel node lymphocytes: tumour reactive lymphocytes identified intraoperatively for the use in immunotherapy of colon cancer

The sentinel node is the first lymph node to receive lymphatic drainage from a tumour and is usually the first site of metastases. Today, the sentinel node is used for tumour staging. Here, we focus on its immunological role and investigate lymphocytic function in sentinel nodes, identified intraoperatively by peritumoural dye injection, from 15 patients with colon cancer. Tumour infiltrating lymphocytes, sentinel and nonsentinel lymph node cells and peripheral blood leukocytes were studied by flow cytometry, proliferation assays and interferon-γ secretion after activation with autologous tumour homogenate. Whereas tumour-infiltrating lymphocytes were nonresponsive in the proliferation assays, lymphocytes from sentinel nodes proliferated dose dependently and secreted interferon-γ upon stimulation with tumour homogenate. The responses were of varying magnitude and tended to be weaker in metastatic sentinel nodes. Sentinel node lymphocytes represents an enriched source of tumour reactive lymphocytes, and may be useful in future trials of adoptive immunotherapy.

Multiplex B Cell Characterization in Blood, Lymph Nodes, and Tumors from Patients with Malignancies

B lymphocytes contribute to immune surveillance, by tumor-specific Abs and Ag presentation to T lymphocytes, but are insufficiently studied in humans. In this article, we report a flow cytometric investigation of B lymphocyte subpopulations in blood, lymph nodes (LNs), and malignant tissues from 20 patients operated on because of advanced solid tumors. The CD19+ compartment in peripheral blood was essentially unaltered in patients, as compared with healthy control subjects. In metastatic LNs, signs of B lymphocyte activation were observed, as evidenced by increased proportions of plasmablasts and CD86-expressing cells. In tumor-infiltrating B lymphocytes (TIL-B), both switched memory cells and plasmablasts were expanded, as compared with nonmalignant epithelium. Moreover, pronounced skewing of Igλ/Igκ ratio was evident among TIL-Bs. By spectratype analysis on IgH, we confirmed a monoclonal expansion of the Vh7 family in TIL-B, also present in a tumor-associated LN. Sequencing the clonally expanded Vh7 revealed signs of somatic hypermutation. In conclusion, B lymphocytes in cancer patients exhibit signs of activation in tumor-associated tissues, likely induced by recognition of tumor Ags. Increased numbers of switched memory cells and plasmablasts in combination with clonal expansion and signs of somatic hypermutation suggest a CD4+ T lymphocyte–dependent antitumoral response, which may be exploited for immunotherapy.

Feasibility of T-Cell-Based Adoptive Immunotherapy in the First 12 Patients with Advanced Urothelial Urinary Bladder Cancer. Preliminary Data on a New Immunologic Treatment Based on the Sentinel Node Concept

BACKGROUND Expected 2-yr survival for patients with urothelial urinary bladder cancer (UBC) with lymph node involvement (pN2) is 20%, regardless of standard neoadjuvant/adjuvant oncologic treatment. Tumor-reactive lymphocytes are present in sentinel nodes (SNs) draining human bladder cancer and display immunologic function on restimulation in vitro. Metinel nodes (MNs) drain secondarily from metastatic tumors and also possess tumor-reactive lymphocytes, which might be a source for adoptive T-cell immunotherapy. OBJECTIVES To determine if MN detection and subsequent expansion of autologous T-helper cells with subsequent reinfusion was feasible and safe to perform in patients with metastatic UBC. DESIGN, SETTING, AND PARTICIPANTS In an open trial, the first 12 included patients are described. Patients were prospectively selected from a single tertiary academic center and had metastatic UBC. All 12 patients were preoperatively staged as T2-T4b N1-2 and/or M0-M1 or MX. INTERVENTIONS MNs were excised in conjunction with intended cystectomy. T lymphocytes were extracted with enhancement and expansion of tumor specific T-helper cells, followed by reinfusion of expanded T cells. MEASUREMENTS All patients were preoperatively staged with transurethral resection of the bladder and routine computed tomography scan. Intended detection of MNs was performed intraoperatively with intended cystectomy. Harvested T cells were evaluated and cell cultures were established. Assessment of reinfusion of expanded, autologous, tumor-specific T-helper cells to six of the patients was performed, focusing on adverse effects. RESULTS AND LIMITATIONS In six patients, it was feasible to administer the treatment. Reinfusion of these T cells was performed without any major adverse effects. In six other patients, we encountered technical failures. CONCLUSIONS A novel adoptive immunotherapy based on T cells from tumor-draining lymph nodes is feasible in advanced UBC. Infusion of expanded, autologous, tumor-specific T-helper cells might be a future treatment option in metastasized UBC. Long-term overall survival remains to be determined.

Evaluation of T and B lymphocyte function in clinical practice using a flow cytometry based proliferation assay

The golden standard for functional evaluation of immunodeficiencies is the incorporation of [(3)H]-thymidine in a proliferation assay stimulated with mitogens. Recently developed whole blood proliferation assays have the advantage of parallel lymphocyte lineage analysis and in addition provide a non-radioactive alternative. Here we evaluate the Flow-cytometric Assay for Specific Cell-mediated Immune-response in Activated whole blood (FASCIA) in a comparison with [(3)H]-thymidine incorporation in four patients with severe combined immunodeficiency. The threshold for the minimum number of lymphocytes required for reliable responses in FASCIA is determined together with reference values from 100 healthy donors when stimulated with mitogens as well as antigen specific stimuli. Finally, responses against PWM and SEA+SEB stimuli are conducted with clinically relevant immunomodulatory compounds. We conclude that FASCIA is a rapid, stable and sensitive functional whole blood assay that requires small amounts of whole blood that can be used for reliable assessment of lymphocyte reactivity in patients.