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The study of regulatory T cells (Treg) requires methods for both in vivo and in vitro analyses, both of which have different limitations, but which complement each other to give a more complete picture of physiological function. Our analyses have focused on Treg-mediated suppression of CD8+ T cells, and in particular Tregs induced by viral infection. One of the unique characteristics of virus-induced Tregs is that they can suppress in vitro without the requirement for additional stimulation. This ability correlates with their activated status in vivo. Furthermore, in vivo activated Tregs can suppress the function of CD8+ T cells both in vitro and in vivo, while leaving proliferation intact. Interestingly, further in vitro stimulation of these Tregs confers to them the ability to suppress both the function and proliferative ability of CD8+ T cell targets.
The model system we use for the study of virus-induced Tregs is Friend virus (FV) infection of adult immunocompetent mice (6). FV is an oncogenic mouse retrovirus that induces acute infections leading to lethal leukemia in most strains of mice (5). However, some strains of mice recover from acute infection, but remain chronically infected for life (4). It is these chronically infected mice that have revealed a role for Tregs in suppressing CD8+ T cells (2).
Interestingly, depletion of CD8+ T cells during acute infection abolishes the ability of high recovery strains of mice to prevent leukemia (3), but depletion during the chronic phase has relatively little effect (4). This finding suggested that chronic FV had escaped CD8+ T cell control. Adoptive transfer studies then showed that chronically infected mice had defective mixed lymphocyte reactions in vivo, and also a decreased CD8+ T cell-mediated rejection of FV-induced tumors in vivo (7). These data suggested a change in T cell function rather than in the virus. Interestingly, further experiments showed that suppression of in vivo CD8+ T cell responses could be adoptively transferred to naïve mice with CD4+ T cells, but not CD8+ T cells, from chronically infected mice. Analysis of the CD4+ T cells revealed that CD25+ T cells were significantly more activated in chronically infected mice than in naïve mice, the same subset of cells that Shimon Sakaguchi had shown to be involved in suppressing anti-self reactivity to prevent autoimmune diseases (10). These studies led to the development of in vivo and in vitro analysis techniques to further study the suppressive activity of virus-induced Tregs (2, 8, 9).
Alternative materials are given in 2.1.8 that will be used depending on the type of assay to be performed. Typically both the target cells and the Tregs are stimulated with anti-CD3-coated plates (11, 12). In such co-cultures cell division of target cells is suppressed. In some situations, such as the study of virus-induced Tregs, it may be of interest to determine the suppressive capacity of the Tregs directly ex vivo, without further stimulation. In such cases rather than stimulating with anti-CD3, which would also activate the Tregs, the target cells may be stimulated with specific peptides, especially if TCR transgenic cells are used as targets.
Suppression of target cells in vivo may be followed using adoptive transfer of labeled cells (see note 5). For example, in Friend virus infections there is a burst of activated Tregs at 2 weeks post-infection (13). By adoptively transferring labeled CD8+ T cells into infected mice around the time of this burst, the effects of suppression on the transferred cells may be observed (2). In addition, adoptive transfer of Tregs from infected mice into naïve mice can be done to monitor their effects in, for example, a naïve mouse (7). We also use this technique to activate CD8+ T cells during acute infections before Treg activity begins. These physiologically activated cells can then be recovered for use in in vitro suppression assays (9).
This research was supported by the Division of Intramural Research of the National Institutes of Health, National Institute of Allergy and Infectious Diseases.
1CFSE concentrations between 2 and 10 μM can be used to adjust the brightness of the labeled cells. Cells used for in vivo transfers will often lose a significant amount of label in vivo so concentrations at the higher end should be used.
2Using CD25 expression to purify Tregs has disadvantages because even in the spleen there are CD25lo Foxp3+ Tregs that will not be acquired using MACS beads or cell sorting using CD25 as the marker. This is especially problematic when purifying Tregs from non-lymphoid tissues, like the liver and gut, where the majority are CD25lo and cannot be purified by these processes. The use of Foxp3-GFP reporter mice is necessary when obtaining CD25lo Tregs from a non-lymphoid tissue or to get the total Treg population from the spleen. In this way, you can stain with anti-CD4 and by FACS cell sorting obtain >95% pure CD4+GFP+ Tregs.
3Remember to not stain cells with FITC or other fluorochromes that are detected in the CFSE channel.
4For lower cell numbers use a 96 well round bottom plate to maximize cell-to-cell contact. Also centrifuge for 2 minutes at 500 rpm after the cultures are set up. Lower cell numbers in the well required a longer in vitro culture (72 hr) because the targets were slower to proliferate and upregulate effector molecules.
5Donor cells can be followed by genetic markers such as Thy1, CD45, or expression of GFP. However, donor cells expressing GFP may be rejected as foreign in experiments lasting more than a week. CFSE-labeled cells can also be used but will lose signal following cells division. They can be followed for at least one month if they do not divide (see note 1).
6The cell concentration will vary depending on numerous variables such as whether the cells will divide, where they will home, how long they will be left in the animal, etc. We have had success with adoptive transfers of as few as 50 cells to as many as 5 × 107. It should be noted that using high numbers of cells may give results not reflective of the true in vivo situation.
7CD8+ T cell function typically stops following harvest and in vitro culture unless the cells are kept stimulated so some type if stimulation is usually required.
8Using a volume of 0.5 ml will help assure that the needle is in a vein and not in tissue. If the needle is in a vein the suspension should flow with very little pressure and should not distend the surrounding tissue. Better results may be obtained using the forefinger rather than the thumb on the plunger of the syringe. The retro-orbital sinus is a convenient site to do intravenous inoculations. Inclusion of heparin sodium in the injection solution is key for the prevention of clotting and pulmonary embolisms that will rapidly kill the recipient mice.