The described experiments indicate that a relatively simple procedure of delivering peptide for a prolonged time period in subimmunogenic forms is suited to induce de novo CD4+25+ suppressor T cells from naive T cells in peripheral lymphoid organs in the absence of a functioning thymus as well as in the absence of a developing immune response. By all investigated criteria (i.e., surface phenotype, long-term stability in the absence of the inducing TCR ligand, Foxp3 expression, Ag-induced expansion in vivo, and suppressive activity in vitro and in vivo as well as cytokine production), the induced suppressor T cells are indistinguishable from intrathymically generated CD4+25+ T cells that were shown to have an essential role in preventing autoimmunity under physiological conditions.
The described experiments are akin to studies conducted by i.v. injection of soluble proteins that were described decades ago and that became known as “low zone tolerance” experiments (29
). It is well possible that, also under those condition, tolerance was due to the induction of suppressor cells as might also be the case in desensitization attempts that have the goal to reduce allergic reactions by applying relevant protein derivatives.
In vivo and ex vivo inductions of suppressor T cells by chronic antigenic stimulation was achieved by several groups (7
), but gave rise to regulatory T cell subsets that appear to be distinct from natural CD25+
suppressor cells. Some in vivo protocols made use of chemicals that affect the entire immune system and, thus, may not be ideally suited to induce Ag-specific unresponsiveness (36
). Ex vivo generation of Ag-specific CD25+
suppressor T cells with IL-2 and TGF-β that resemble naturally occurring suppressor T cells has been reported previously (36
), but little is known about the long-term survival of these cells in vivo in the absence of Ag. In one particular setting of suppressor T cell induction in vivo, it was found that CD4+
anergic cells could accumulate after i.v. or oral administration of Ag, but Foxp3
expression was not analyzed (25
). Whether this resulted from de novo generation of suppressor cells or their selection was not entirely clear. Also, this particular protocol appeared to induce a transient immune response that was not observed after peptide infusion.
The mechanism by which the peripheral differentiation of naive T cells into CD25+
Ag-specific suppressor cells that resemble by all criteria natural suppressor cells as reported in the present work remains to be defined. It is possible that this process could be initiated by interactions with HA peptide–presenting nonactivated DCs such as epidermal Langerhans cells, which are at the site of osmotic pump implant, can endocytose efficiently, and are endowed with trafficking capacity to secondary lymphoid tissues where they can induce tolerance in the steady state (38
). Thus far, it has been demonstrated that Ag presentation by nonactivated DCs can result in recessive tolerance (40
), but it is possible that low doses of peptide presented over relatively long time periods (10 d) on nonactivated DCs represents a natural way of inducing active immunosuppression by which the immune system prevents immune responses to self components that do not induce tolerance in the thymus. Such a scenario may also lead to the suppression of antitumor responses when tumor-specific peptides are presented in subimmunogenic form. At present, it is not entirely clear how the immune system can evade this suppression, but strong activation of APCs may be one mechanism (42
The fact that naive CD25−
T cells can be instructed in vivo, as reported here, or in vitro (13
) to become Foxp3
suppressor cells raises the question of whether naturally occurring Foxp3
suppressor T cells that develop in the thymus constitute a lineage distinct from that of their peripherally induced counterparts, or whether such cells reflect the existence of a single Foxp3
-expressing naturally occurring CD25+
suppressor lineage that can develop under noninflammatory steady state conditions in primary as well as secondary lymphoid tissues.
It needs to be tested whether the procedure described here will be suited to induce prospective tolerance to a large variety of Ags, including transplantation Ags. By means of single peptides, efficient suppression could be achieved because suppressor cells cannot only suppress responses of T cells with the same Ag specificity but also responses of neighboring T cells that happen to interact with other ligands in the vicinity of suppressor T cells (bystander suppression). Thus, it will be of interest to monitor whether, eventually, the in vivo induction of Ag-specific suppressor cells by the described procedure will make general immune suppression the therapy of the past.