Antigen-bearing immature dendritic cells induce peptide-specific CD8⁺ regulatory T cells in vivo in humans

Regulatory T cells (T_Rs) have now been clearly identified in mice and humans.1-3 These cells can inhibit strong responses mediated by CD4⁺ and CD8⁺ effector T cells, thus preventing allograft rejection, graft-versus-host disease (GVHD), chronic inflammatory disease, and autoimmunity (for a review, see Roncarolo and Levings,1Waldmann and Cobbold,2 and Sakaguchi3). Recent studies have identified T_Rs in human blood, where they have 2 main functional properties4-6: they proliferate poorly in response to mitogenic stimuli and they can dampen the responses of effector T cells.7 Although most studies have characterized CD4⁺ T_Rs,8,9CD8⁺ T cells with regulatory properties have also been described.10-16 

Certain populations of T_Rs, particularly those expressing CD4 and the CD25 interleukin 2 (IL-2)–receptor chain, are generated in the thymus, where the cortical epithelium has been identified as a critical antigen-presenting cell (APC).17T_Rs, often identified by their capacity to produce IL-10, can also be induced peripherally in the settings of transplantation and GVHD,1 2 but the APC requirements have not been identified. It is important to identify pathways that control the formation of T_Rs, since these would provide novel strategies for antigen-specific immune suppression or immune tolerance.

Dendritic cells (DCs) are powerful APCs for the induction of effector T cells.18 To initiate immunity, DCs must carry out 2 sets of linked events.19 One is the capture of antigens and successful formation of major histocompatibility complex (MHC)–peptide complexes; the second is to undergo a process termed “maturation” to acquire many additional properties to stimulate immunity.19 Immature DCs appear to be inactive as inducers of immunity in vivo.20 However, in a standard tissue-culture assay involving initiation of the mixed leukocyte reaction, immature DCs were not inactive but instead induced the formation of T_Rs, with both the anergic and regulatory properties mentioned above.21 

In parallel, we tested the effects of immunizing volunteers with immature DCs. We reported findings in 2 healthy volunteers who received a single subcutaneous injection of 2 × 10⁶ immature DCs pulsed with an HLA-A*0201–restricted influenza matrix peptide (MP).22 In contrast to previous findings using mature DCs,23 injection of immature DCs was associated with antigen-specific inhibition of effector T-cell function. The peptide-specific, interferon-γ (IFN-γ)–producing cells disappeared from the blood, and cytolytic cells could no longer be expanded in culture. However, antigen-binding CD8⁺ T cells were still present, and circulating MP-specific IL-10 producers developed. In summary, the use of immature DCs silenced effector T-cell functions, raising the possibility that T_Rs were being induced in vivo.

In the study described here, we showed that the loss of effector function and induction of IL-10 producers is self-limited, since there was a return to preimmunization status by 6 months after immunization. Importantly, we found that 1 week after immunization, the blood did contain CD8⁺ T cells with T_R function, able to block the function of CD8⁺ effectors that secrete IFN-γ. These data provide evidence that immature DCs can induce antigen-specific CD8⁺ T_Rs in vivo in humans.

This report describes the findings in 2 volunteer subjects (Im1 and Im2) who received subcutaneous injections of immature DCs derived by culture of blood monocyte precursors in granulocyte-macrophage colony-stimulating factor and IL-4 as described previously.22 The injected DCs were pulsed with keyhole-limpet hemocyanin and influenza MP during the last 16 hours of a 6-day (Im1) or 7-day (Im2) monocyte culture as described previously.22 Approval was obtained from the institutional review board for these studies. Informed consent was provided according to the Declaration of Helsinki.

Both subjects were evaluated 1 week after DC injection and at 1- to 3-month intervals afterward. Both had normal results on repeated hemogram assessments and no rheumatoid factor or antinuclear antibody 1 and 3 months after DC injection. Antigen-specific T cells were quantified by using a standard enzyme-linked immunospot (ELISPOT) assay for the presence of peptide-specific cells producing IFN-γ, IL-4, or IL-10.22 For cytolytic T-lymphocyte (CTL) assays, T cells were cocultured with peptide-pulsed mature DCs for a week, before measurement of lytic activity, as described previously.22DC maturation was achieved by day 1 of culture in a mixture of IL-1β, IL-6, tumor necrosis factor α, and prostaglandin E₂.

Peripheral blood mononuclear cells (PBMCs) obtained 7 days after immunization (T_R sample), before immunization, or at recovery (eg, day 180) were thawed and cultured (2-3 × 10⁵ cells/well) either separately or together in the presence of peptide-pulsed, autologous monocyte-derived mature DCs at a PBMC-to-DC ratio of 60:1. Antigen-specific INF-γ–producing cells were quantified by using a standard ELISPOT assay as described previously.23 In addition to the immunizing peptide (influenza MP; GILGFVFTL), additional control HLA-A*0201–restricted peptides were from Epstein-Barr virus latent membrane protein 2 (EBV LMP-2; CLGGLLTMV) and human immunodeficiency virus 1 (HIV-1) gag (SLYNTVATL).

For some experiments, T_Rs containing PBMCs (from day-7 samples) were depleted of CD8⁺ T cells with use of immunomagnetic beads (Miltenyi Biotec, Auburn, CA) before they were added to the cocultures. For some experiments, the T_Rsamples were separated from the recovery specimens by a transwell to check for soluble suppressor factors. In these cultures, APCs were added on either side of the transwell. In some experiments, the cocultures of T_Rs and recovery cells were performed in the presence of neutralizing anti–IL-10 antibody (10 μg/mL; R&D Systems, Minneapolis, MN) or 100 U/mL recombinant IL-2 (rIL-2; Chiron, Emeryville, CA).

The Student t test was used to compare results in different groups. The significance level was set atP < .05.

Both healthy volunteers had been primed to influenza at baseline because influenza MP–specific effector T cells were detectable on ELISPOT assays and peptide-specific CTLs could be expanded by a week of culture with mature DCs. However, 1 week after the injection of MP-pulsed immature DCs, these effector functions in the blood were silenced. This loss of function was reversible, with values returning to preinjection levels by 3 to 4 months after injection in both subjects (Figure 1). In a reciprocal fashion, silencing and recovery of effector T-cell function were associated with the appearance and then decline in peptide-specific IL-10 producers, which were no longer detectable after 90 to 100 days postimmunization (Figure 1). The DC injections were not associated with any clinical toxicity or clinical or serologic evidence of autoimmunity in either subject. Thus, the inhibition of effector T-cell function after a single injection of immature DCs was found to be self-limited.

Because we had previously shown that the loss of circulating MP-specific effector T-cell function was not associated with a decline in circulating MHC tetramer–binding cells,22 we tested whether the effector silencing after injection of immature MP-pulsed DCs was mediated by the induction of T_Rs. To assess this directly, we mixed T cells from samples obtained 1 week after immunization (when the effector silencing was maximal) with samples obtained before immunization. The PBMCs obtained on day 7 inhibited MP-specific producers of INF-γ from cultures of preimmunization samples from both subjects (Figure 2A). The inhibition was specific for the immunizing peptide because responses to the control peptide LMP-2 were not silenced.

Further characterization of the suppression was carried out only in samples from Im2, from whom we had additional cells available. The suppression of T-cell function was not due simply to competition for APCs or consumption of IL-2, since it was specific for immunizing peptide and observed only when suppressor samples (day 7) but not nonsuppressor (preimmunization) samples were added to the recovery samples (day 180; Figure 2B). T-cell suppression was dose dependent and observed even when the ratio of day-7 to day-180 cells was 1:10. Suppression was lost if day-7 cells were depleted of CD8⁺ T cells or if cell contact between day-7 and recovery T cells was prevented in transwell cultures (Figure 2C). Although the day-7 specimens had been shown to contain MP-specific IL-10 producers, addition of neutralizing anti–IL-10 antibody led to only a slight recovery of MP-specific effectors. However, the suppression was fully reversed by addition of 100 U/mL rIL-2. Thus, we found that peptide-specific CD8⁺ T_Rs induced in vivo by immature DCs inhibit CD8⁺ T cells in a cell-contact–dependent manner, that is, a manner largely independent of IL-10.

These data provide direct evidence for the existence of antigen-specific CD8⁺ T-cell–mediated immune regulation and of the induction of such T cells in vivo in humans by immature DCs. Once induced, these cells have a limited life span in the circulation. Thus, naturally occurring T_Rs may require continued antigen presentation by trafficking immature DCs. Because peptide-specific, IL-10–producing cells are also induced by immature DCs, we refer to these suppressor cells as T_Rs, in keeping with previously established nomenclature. The regulation we observed required cell-cell contact and was largely independent of IL-10. These features are similar to those of CD4⁺ T_Rs induced by immature DCs in vitro.21 A subset of CD8⁺CD28⁻ suppressor T cells that mediate suppression in a cell-contact–dependent fashion has also been described.12-14 

The site where immature DCs generate T_Rs in vivo is not known. One possibility is that the DCs might traffic to lymph nodes to meet T cells recirculating by means of high endothelial venules. An alternative, which we favor because T_Rs have an activated phenotype, is that the DCs activate T_Rs that circulate from blood to extravascular spaces (here, the skin) and then return to the lymph node by means of the lymphatics. Although our studies help to confirm the presence of T_Rs and IL-10–producing cells in the CD8 compartment, compared with previously described CD4⁺ T_Rs, further work is needed to clarify their relation to CD25⁺ suppressor cells and their mechanism of action.

Our data suggest DC maturation as a key therapeutic target for the regulation of immunity.19 The inhibition of maturation in antigen-capturing DCs may promote the induction of T_Rs in vivo. Impairment of CD8⁺ T-cell suppressor function has been observed in patients with human autoimmune diseases such as lupus and multiple sclerosis.15,16 A role for T_Rs in acceptance of human allografts has also been suggested.24 In a reciprocal fashion, reduction of T_Rs may improve resistance to cancer and chronic infections, as was observed in a study of experimental tumors in mice.25 

We thank Joseph Krasovsky for excellent technical assistance.