Regulatory T cells : molecular and clinical aspects

In the immune system’s tug of war with cancer, tolerance mechanisms by which the tumor can control anti-tumor immune responses play a central role in determining the outcome. Regulatory T cells (Treg) induced in the thymus or the periphery, represent one such tolerance mechanism that potentially can be exploited by developing tumors. In this thesis, we explore the underlying molecular mechanisms that result in Treg development, and attempt to elucidate the importance of this T lymphocyte subset in urinary bladder cancer. In humans and mice, the transcription factor FOXP3 is crucial for the function of the Treg subset and maintenance of peripheral tolerance, as illustrated by the lack of functional Treg and autoimmune disorders that result from mutations within this protein. In humans however, clinical studies of this important T lymphocyte subset are obscured by the fact that FOXP3 is transiently induced in conventional T lymphocytes upon activation. Initially, we address the epigenetic regulation of FOXP3 expression, and demonstrate that the committed Treg population has an almost completely demethylated FOXP3 promoter region, whereas conventional non-regulatory CD4+ T lymphocytes are semi-methylated in this region. Furthermore, we study the development of regulatory T cells in mice, and assess the contribution and the impact of adenosine receptor signaling on the T lymphocyte compartment. Next, the role of the Treg population in urinary bladder cancer is evaluated. First, we investigate the impact of tumor infiltrating CD3+ as well as FOXP3+ T lymphocytes on patient survival. Somewhat surprisingly, we find a positive correlation between both CD3+ and FOXP3+ lymphocytic infiltrates, suggesting that FOXP3+ lymphocytes in this case may not represent a tumor escape mechanism as initially hypothesized. In addition, we observe FOXP3 expression in a subset of tumors, and find that this expression is a negative prognostic factor for survival. To follow up these results, we characterize the T lymphocyte immune response in peripheral blood, lymph nodes and tumor tissue from patients with UBC. We demonstrate that the FOXP3+ fraction of CD4+ T lymphocytes is significantly increased compared to all other locations investigated including macroscopically healthy bladder tissue. Furthermore, these tumor infiltrating lymphocytes express high levels of activation and effector markers, but do not display a demethylated pattern in the FOXP3 promoter to match its prominent expression. Interestingly, muscle invasive tumors have a lower FOXP3+ fraction at the invasive front compared to non-invasive counterparts. In addition, we observe changes in the cellular immune response dependent on if the patients have received neo-adjuvant chemotherapy or not, both with regard to cell composition and functional reactivity to tumor antigens. Epigenetic regulation governs the commitment of T lymphocytes to the Treg lineage. The fact that FOXP3 expressing tumor infiltrating lymphocytes in UBC do not display a committed Treg phenotype could potentially explain the differences in reported clinical impact of this population in different cancers and has implications for future immunotherapy

Long Lasting Local and Systemic Inflammation after Cerebral Hypoxic ischemia in Newborn Mice

Background: Hypoxic ischemia (HI) is an important cause of neonatal brain injury and subsequent inflammation affects neurological outcome. In this study we performed investigations of systemic and local activation states of inflammatory cells from innate and adaptive immunity at different time points after neonatal HI brain injury in mice. Methodology/Principal Findings: We developed a multiplex flow cytometry based method combined with immunohistochemistry to investigate cellular immune responses in the brain 24 h to 7 months after HI brain injury. In addition, functional studies of ex vivo splenocytes after cerebral hypoxic ischemia were performed. Both central and peripheral activation of CD11b + and CD11c + antigen presenting cells were seen with expression of the costimulatory molecule CD86 and MHC-II, indicating active antigen presentation in the damaged hemisphere and in the spleen. After one week, naïve CD45rb + T-lymphocytes were demonstrated in the damaged brain hemisphere. In a second phase after three months, pronounced activation of CD45rb 2 T-lymphocytes expressing CD69 and CD25 was seen in the damaged hemisphere. Brain homogenate induced proliferation in splenocytes after HI but not in controls. Conclusions/Significance: Our findings demonstrate activation of both local and systemic immune responses months after hypoxic ischemic neonatal brain injury. The long term immune activation observed is of general importance for future studies of the inflammatory response after brain injury as most previous studies have focused on the first few weeks afte

LPS Regulates SOCS2 Transcription in a Type I Interferon Dependent Autocrine-Paracrine Loop

Recent studies suggest that SOCS2 is involved in the regulation of TLR signaling. In this study, we found that the expression of SOCS2 is regulated in human monocyte-derived DC by ligands stimulating TLR2, 3, 4, 5, 8 and 9 signaling. SOCS2 induction by LPS was dependent on the type I IFN regulated transcription factors IRF1 and IRF3 as shown by using silencing RNAs for IRFs. Blocking endogenous type I IFN signaling, by neutralizing antibodies to the receptor IFNAR2, abolished SOCS2 mRNA expression after TLR4 stimulation. Transcription factors STAT3, 5 and 6 displayed putative binding sites in the promoter regions of the human SOCS2 gene. Subsequent silencing experiments further supported that STAT3 and STAT5 are involved in LPS induced SOCS2 regulation. In mice we show that SOCS2 mRNA induction is 45% lower in bone marrow derived macrophages derived from MyD88−/− mice, and do not increase in BMMs from IRF3−/− mice after BCG infection. In conclusion, our results suggest that TLR4 signaling indirectly increases SOCS2 in late phase mainly via the production of endogenous type I IFN, and that subsequent IFN receptor signaling activates SOCS2 via STAT3 and STAT5

Single Dose Caffeine Protects the Neonatal Mouse Brain against Hypoxia Ischemia

<div><p>In this randomized blinded study, we investigated caffeine 5 mg/kg treatment given directly after neonatal brain hypoxia ischemia. Brain morphology, behavior and key brain infiltrating immune populations were examined. Caffeine treatment significantly improves outcome when compared to phosphate buffered saline. Flow cytometric analysis of immune responses revealed no persistent immunological alterations. Given its safety caffeine emerges as a candidate for neuroprotective intervention after neonatal brain injury.</p></div

Heat map of all investigated immune populations after sham operation or HI 24h, 72h and two weeks after randomization to caffeine or PBS (n = 3–5 in each group, total n = 52).

<p>The data was normalized to zero for the lowest value and one for the highest value in each variable with zeros in blue through yellow to ones values in red. Whites are missing values. MFI = Median florescence intensity.</p

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(+)CD25(hi) Tregs displayed a demethylated FOXP3 promoter (1.4%+/-0.95% SEM methylated) in contrast to CD4(+)CD25(lo) T cells which were partially methylated (27.9%+/-7.1%). Furthermore, stimulated CD4(+)CD25(lo) 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-beta 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

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

IRF3, but not MyD88, mainly involved in SOCS2 mRNA expression after BCG infection in mice bone marrow macrophages (BMM).

<p>BMM from wild-type mice, MyD88 ^−/− mice or IRF3 ^−/− mice were activated with BCG for indicated time periods, the cells were then harvested for SOCS2 mRNA measurement by qRT-PCR. Data shown are the mean of triplicate determinations from three independent experiments. Values for time point 0 were set to 1. Significance comparing results from the deficient mice to wild-type mice is indicated (* p<, 0.05; ** p<, 0.01).</p