Analysing the Phenotype and Function of Regulatory T Cells

EuroSciCon'ss focus on novel methods and their applications was at the fore in this informative meeting on April 4th covering some of the latest findings on regulatory T cells. The popularity of this field was reflected by the number of delegates with a lively audience of 150 research scientists filling the Birkbeck lecture theatre in London UK. Talks covered the methodology of regulatory T cell isolation, quantification and expansion and data were presented from the fields of reproductive immunology, autoimmunity, inflammation (collitis) and transplantation.

The day was started with a talk by Dr Ana Claudia Zenclussen (Institute of Medical Immunology, Germany) who introduced the topic by covering the range of T cells that come under the umbrella title of regulatory T cell subsets. These include natural CD4+CD25+ thymus derived T cells, and their induced CD4+CD25+ counterpart, CD4 Tr1 cells, CD4+ Th3/Tr2 and CD8+ Tr1 or Tr2. Many research groups focus their work on the first two groups and we were reminded that using CD25+ as the principal marker of these cells could be inaccurate as this was also a marker of recently activated effector T cells. Obviously a key feature of this field is the ability to distinguish regulatory T cells from other T cells with markers other than CD25, CTLA-4 etc., and it was pointed out that several novel markers have been identified in recent years such as neuropilin-1 and FoxP3 (the forkhead-winged helix family transcription factor that controls differentiation of natural CD25+ regulatory T cells). FoxP3 is also on other T cells, but its levels are 100x more on regulatory T cells. In general, in this report, regulatory CD4+ T cells as determined by high CD25 expression are described as Tregs unless qualified further.

Dr Zenclussen went on to describe a model of spontaneous abortion where a CBA/J(H2k) female crossed with a Balb/c(H2d) male results in a normal pregnancy, whereas a CBA/J(H2k) female crossed with a DBA/2J(H2d) male results in an abortion prone female. In these latter mice, there is an increase in Th1 IFN gamma producing male antigen specific T cells at the materno-foetal interface and this appears to be associated with a downregulation in Treg activity, both in terms of function and foxP3 and neuropilin expression. Transfer of Tregs from pregnant (but not non-pregnant) normal mice prevents rejection of the foetus, and is associated with an increase in TGF-beta expession at the materno-foetal interface. Tregs are generated very early in pregnancy, in a male antigen specific manner but independantly of hormones, and can be detected at day 2 in draining lymph nodes and vaginal mucus.

This talk was followed by several covering the field of autoimmunity. Dr Lucy Walker (University of Birmingham) started by reminding the audience of one of the central issues in autoimmunity, living with self reactivity. She'ss using a transgenic model of diabetes where the antigen ovalbumin is expressed under the control of the insulin promoter combined with OVA specific DO11.10 T cells. Dr Walker has been trying to determine the changes in relative numbers of pathogenic T cells and Tregs during the course of disease, the migration of Tregs and the phenotype of Tregs when they'sre actively regulating. In particular, how do you measure the activation status of cells which are already bearing activation makers As determined by FoxP3 staining, Tregs accumulate in the pancreatic lymph nodes. Treg acculumation here is antigen specific, which begs the question is there local activation of Tregs in the pancreatic lymph nodes Dr Walker showed that CD69 expression precedes Treg acculumation with expression on 50-60% of Tregs in the pancreatic lymph nodes versus 5-10% in the spleen. In the presence of Tregs, there is decreased infiltration of pathogenic T cells into the pancreatic islets. In addition, Tregs decreased the production of IFN-gamma by DO11.10 specific T cells. Professor David Wraith (University of Bristol) continued the theme of autoimmunity, presenting some of his groups results with the animal model of multiple sclerosis and relating this to others in the field of peptide-induced tolerance. Injection, or nasal adminstration, of soluble peptides such as Ac1-9 (the N terminal epitope from myelin basic protein) can induce a distinct poplulation of Tregs that are anergic and IL-10 dependent. Peptide-induction of Tregs occurs even in the absence of natural Tregs. Administration of anti-IL10R antibody reverses suppression of proliferation in vivo. This type of peptide-induced tolerance is antigen-specifc in the induction phase and involves bystander suppression in the effector phase. Interestingly peptide-induced Tregs are 90%CD25- and 10% CD25+. This latter sub-population are not required for in vitro suppression by peptide-induced Tregs. FoxP3 is not present on peptide-induced Tregs. Human peptide therapy trials are now been carried out in allergies (bee venom and cat dander), rheumatoid arthritis, type I diabetes and multiple sclerosis (including the Bristol group). Effects seen to date have involved regulation by IL-10 producing T cells and Th cell deviation.

Dr Juergen Hass (University of Heidelberg, Germany) has been studying the effect of therapy with immunomodulatory drugs such as IFN-beta and the polymer glatiramer acetate (GA) in patients with multiple sclerosis. Both of these drugs appear to reduce the risk of further relapse, appearance of new lesions and the progression of disability. Multiple sclerosis patients are know to have impaired Treg function (although Treg frequencies are similar to normal individuals), but after treatment with both these drugs there was an increase in the suppressive function of the Tregs. In co-culture experiments, IFN-beta had a direct effect on Tregs. After 18 hours pre-exposure, both drugs decreased the production of Th1 cytokines and increased Th2 cytokine production in effector T cells. IFN-beta, but not GA, reduced T cell proliferation. Dr Michael Ehrenstein (UCL, London) continued the theme of Tregs in the clinical setting, namely those patients with rheumatoid arthritis. He pointed out that much of the work on Tregs has been done in animal models. For example, depletion of Tregs in collagen-induced arthritis increases the incidence and severity of disease. However, a smattering of publications are now emerging in the field of human rheumatoid arthritis. Like in multiple sclerosis, he has shown that the numbers of CD4+ CD25hi cells are similar in nomal individuals and patients. Tregs from rheumatoid arthritis patients are anergic and suppress proliferation of effector T cells, but not pro-inflammatory cytokine production by T cells and monocytes. In patients treated with anti-TNF alpha therapy (Infliximab), the number of Tregs increased and their suppressive activity was restored with a rise in NOS and Foxp3 levels and a decrease in CRP levels. The Tregs in rheumatoid arthritis appear to be generated de novo in the joints. Rheumatoid arthritis was further covered in a talk by Dr Leonie Taams (King'ss College, London), who described her work in patients treated with methotrexate (MTX). She has observed that levels of CD4+ regulatory T cells, in particular the CD25hi subset, are raised in the peripheral blood and synovial fluid of patients versus normals. But, like the previous speaker, they have been shown to be functional defective. This may be due to the inflammatory mileu in which they function. Treatment of rheumatoid arthritis with MTX does not change the frequencies of Tregs, but in synovial fluid they suppress T cell proliferation and production of TNF alpha, IL-10 and IFN gamma more than Tregs from untreated patients or normal controls. The effects of a pro-inflammatory environment were discussed, specifically the activation status of monocytes. Monocytes in the synovial fluid are activated, compared with peripheral blood monocytes from the same patients, with high levels of CD80, CD86 and HLAII. Using a Luminex approach it was shown that CD4+CD25+, but not CD4+CD25- T cells, decreased production of IL-1beta, IL-8 and MIP1alpha by monocytes in vitro. Transwell and co-culture experiments demonstrated that this was a contact dependent effect, with a decrease in monocyte IL-6 production associated with IL-10 production.

The last talk before lunch was given by Dr Ana Izcue (Oxford University) who covered the topic of Tregs in intestinal inflammation. She described a model of collitis induced by transfer of CD45RBhi naive T cells into immunodeficient mice. The disease can be abrogated by cotransfer of CD4+CD25+ Tregs. Both CTLA-4 and IL-10 are involved in this suppression, with the former having a more systemic effect and the latter being important in controlling intestinal pathology. The IL-10 appears to be produced by the progeny of CD4+CD25+ Tregs in the colon, but not spleen or MLN, suggesting that IL-10 may be involved in an intestine specific mechanism for immune regulation. She then went on to discuss the CTLA-4 knockout mice. This costimulatory molecule is transiently expressed on activated T cells and is widely implicated in control of cancer and autoimmunity. CTLA-4 expression is high in CD25+ T cells and anti-CTLA-4 therapy abrogated protection from collitis. Although CTLA-4 knockout mice die after several weeks, this mice does have Foxp3+ T regs. It appears that anti-CTLA-4 reversal of suppression only works if CTLA-4 expression is on CD25+ cells, and that anti-CTLA-4 treatment does not deplete T regs (as determined by Foxp3 expression), but rather blocks the proliferation or development of T regs. CTLA-4 knockout mice have a compensatory mechanism, which may be associated with the high levels of IL-10 found in these mice. Administration of anti-IL10 to these mice results in an increased susceptibity to collitis.

After some animated discussions around lunch and a chat with many of the representatives of the Biotech companies present (including Beckman, Merck Biosciences, LI-COR Biosciences Uk Ltd, Bio-Rad Laboratories Ltd, Synoptics Ltd and GenoLogics ), we heard three talks on the issues related to isolation and expansion of Tregs. Dr Doris Hasselmann (Miltenyi Biotech, Germany) reviewed seperation procedures for Tregs. These involved depletion of CD4- cells followed by positive selection of CD4+CD25+ T cells using magnetic labelling. Optimimising the quantity of CD25 microbeads allows the purification of CD25hi expressing CD4+ T cells, 90% of which are Foxp3+. Cell populations with a purity of approximately 85% can be recovered within 2 hours and the microbeads used are biodegradable. Cells purified by this method have been used successfully in eg. adoptive transfer experiments and co-culture experiments in vitro. Mr Thomas Bauer outlined Invitrogen Corporation'ss approach to isolation and expansion of Tregs. He reminded us that no specific CD marker has been assigned to Tregs and therefore we are still reliant on the isolation of CD4+CD25hi+ T cells, 1-3% of the total CD4+ T cell population. Again negative depletion of non-CD4+ cells was followed by positive selection of CD25+ T cells, captured magnetically. This bead-based system then makes use of a polyclonal anti-Fab (DETACHaBEAD) to detach the cells from the beads. Antibody and bead free Tregs can then be used in a number of applications. To expand this purified T reg population, Dynabeads coated with anti-CD3 and anti-CD28 will give a 1000 fold expansion over 21 days. After magnetic removal of the beads, these expanded Tregs were capable of suppressing an allogenic T cell proliferation in a MLR. Dr Frederique Ponchel (St James's University Hospital, Leeds) highlighted the importance of variation in anti-human CD25 antibody clones, and discused how much individual variation, user error and technical limitations were also responsible for the variations in % cells stained and the intensity of staining observed in the literature. Her group have been testing the staining profiles of five commercially available anti-CD25 monoclonal antibodies, using three FACS machines and analysis with CellQuest and Kolmogorov-Smirnov statistics. Not surprisingly there was some variation in the staining profiles, attributed to the specific antibody, population variability and gating strategy. We await with interest the publication of these results, in particular the details of the antibodies used!

The last three talks of the meeting concentrated on the therapeutic properties, or otherwise, of Tregs. Mr Brendan Meyer (St George'ss Hospital, London) described the use of the thalidomide analogues, CC-407 and lenalidomide, in cancer therapy, specifically in multiple myeloma. The mode of action of the analogues, but not thalidomide itself, appears to be on Tregs, which are known to suppress anti-tumour immunity. The thalidomide analogues suppressed T regulatory cell expansion in IL-2 treated PBMC cultures. Dynal bead selection was used to isolate T regs for in vitro analysis, and these CD4+CD25hi, CTLA-4+, FoxP3+ cells appeared to have reduced suppressive activity against autologous PBMC in the presence of anti-CD3. Dr Halima Moncrieffe (NIMR, Mill Hill, London) works on GVHD, a well recognised complication of allogeneic haematopoietic stem cell transplantation. In her model of GVHD, naive H2b CD4+ CD25+ CD44lo cells are transferred into a F1 cross of Rag2-/- x H2b/d mice. This results in severe GVHD, which can be controlled by co-transfer (with naive T cells) of CD4+CD25+ Tcells or memory CD4+ Tcells. These 2 cell populations differ in their location post-transfer with Tregs migrating to the spleen and MLN, while memory cells are concentrated in the periphery. Intracellular staining revealed that Tregs inhibit IFN gamma production in the naive cell input. OX40 is also a crucial molecule in controlling GVHD with much less severe disease in the OX40 knockout. The final talk was given by Dr Jian-Guo Chai (Imperial College, London) who brought us back to the hurdles of working with a scarce population of cells that are anergic and lack defined specificity. His group have been working, with others, on methods of generating large numbers of Tregs in vitro that can be used in vivo to induce tolerance, in this case in the field of transplantation. For example FoxP3 can be retrovirally transduced into HY-specific TCR transgenic CD4+CD25- mouse T cells. These cells then display a Treg phenotype and function as determined by FACS, T cell proliferation assays, ELISA and intracellular cytokine staining.. In vitro these Tregs were anergic and suppressive and in vivo Treg transfer prevented rejection of male skin grafts by syngeneic females. A question still remains about the ability to generate FoxP3 Tregs from naive CD4+ T cells in the periphery. However, T cell FoxP3 gene transfer seems an interesting candidate for the control of transplantation tolerance.

Overall, this was an interesting meeting that highlights the need for regular updates on how the methodology, both new initiatives and further exploration of established techniques, drives research in the Life Sciences. My thanks go to Shara Cohen and her colleagues at EuroSciCon ( www.euroscicon.com ) for all their hard work in setting up such meetings.

Dr Catherine Derry.

Science Communicator

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