CD40 signalling induces IL-10-producing, tolerogenic dendritic cells.

Dendritic cells (DC) are potent antigen-presenting cells capable to induce efficient antigen-specific T cell responses in vitro and in vivo. Herein, the maturation process is of great significance, as immature DC (iDC) are known to induce rather regulatory than effector T cell differentiation. This study was designed to characterize the role of the CD40-CD40L pathway for differentiation and function of human DC. Therefore, iDC were stimulated through CD40-CD40L interaction by transduction of DC with adenoviral vectors encoding for CD40L (Ad-CD40L). Resulting DC (CD40L-DC) were analysed concerning their phenotype, cytokine profile and T cell stimulatory capacity. Transduction induced a DC phenotype comparable to stimulation with proinflammatory cytokines as revealed by upregulation of CD83 and the costimulatory molecules CD80 and CD86. Additionally, Ad-CD40L-induced strong production of IL-12p70 not observed in cytokine-matured DC. Surprisingly, the T cell stimulatory capacity was markedly reduced in CD40L-DC. Furthermore, stimulation of CD8(+) T cells by peptide-loaded CD40L-DC resulted in a substantial reduction of antigen-specific IFN-gamma production. This phenomenon is due to an enhanced IL-10 production of CD40L-DC in DC-T cell coculture as well as a stabilization of the IL-10 receptor expression on activated T cells. These data demonstrate that DC stimulated through CD40-CD40L interaction differentiate into tolerogenic DC with immunomodulatory function.

Depletion of CD4+CD25+ human regulatory T cells in vivo: kinetics of Treg depletion and alterations in immune functions in vivo and in vitro.

The aim of this study was to investigate whether depletion of CD4(+)CD25(+) regulatory T cells (Treg) from melanoma patients affects immune responses against tumors. By application of recombinant IL-2-diphteria toxin fusion protein, also known as ONTAK, we were able to significantly reduce the frequency of Treg in peripheral blood, whereas other cell populations remained unaffected. The reduction of Treg started immediately after the first bolus of ONTAK with a dose of 5 microg ONTAK per kg bodyweight and lasted for 13 days with subsequent recovery thereafter. Successive ONTAK treatments further reduced the number of circulating Treg. Using the contact sensitizer DCP we show that all patients developed vast eczema after Treg depletion, whereas no or only mild eczematous reactions were detectable before ONTAK treatment. Corresponding induction of DCP-specific CD4(+) and CD8(+) T cells were detectable. Moreover, after immunization of ONTAK treated patients with tumor antigen peptides, MelanA/MART-1 and gp100, significant induction of peptide specific CD8(+) T cells could be observed in 90% of the patients treated. These cells displayed effector functions, as they were able to lyse peptide-pulsed target cells and secreted IFNgamma upon restimulation. In aggregate, our data indicate that ONTAK depletes Treg in vivo significantly, resulting in enhanced immune functions and substantial development of antigen-specific CD8(+) T cells in vaccinated individuals.

Targeting of antigens to activated dendritic cells in vivo cures metastatic melanoma in mice.

Anti (alpha)-DEC-205 antibodies target to the DEC-205 receptor that mediates antigen presentation to T cells by dendritic cells. To exploit these properties for immunization purposes, we conjugated the melanoma antigen tyrosinase-related protein (TRP)-2 to alphaDEC-205 antibodies and immunized mice with these conjugates together with dendritic cell-activating oligonucleotides (CpG). Upon injection of the melanoma cell line B16, alphaDEC-TRP immunized mice were protected against tumor growth. Even more important for clinical applications, we were able to substantially slow the growth of implanted B16 cells by injection of alphaDEC-TRP2 conjugates into tumor bearing hosts. Approximately 70% of the animals were cured from existing tumors by treatment with alphaDEC conjugates carrying two different melanoma antigens (TRP-2 and gp100). This protection was due to induction of melanoma-specific CD4 and CD8 responses. Thus, these data show that targeting of dendritic cells in situ by the means of antibody-antigen conjugates may be a novel way to induce long-lasting antitumor immunity.

Human CD25+ regulatory T cells: two subsets defined by the integrins alpha 4 beta 7 or alpha 4 beta 1 confer distinct suppressive properties upon CD4+ T helper cells.

Down-regulation of autoreactive T cell responses in vivo includes cell-contact-dependent as well as contact-independent mechanisms. Infectious tolerance is a contact-dependent mechanism used by naturally occurring CD25(+) T regulatory cells (Tregs) to confer suppressive activity upon conventional CD4(+) T cells thereby generating secondary T helper suppressor cells(Th(sup)), which inhibit T cell activation via soluble mediators. Here, we describe two distinct subsets of human Tregs, characterized by expression of either the alpha(4)beta(7) integrin or the alpha(4)beta(1) integrin. Upon activation, both subsets show an enhanced expression of FoxP3, recently described as a key transcription factor of murine Tregs. In addition, both are able to convey suppressive capacity to conventional CD4(+) T cells. However, the properties of Treg subsets are rather distinct: alpha(4)beta(7) (+)Tregs induce IL-10-producing Th(sup) (Tr1-like), whereas alpha(4)beta(1) (+) Tregs induce TGF-beta-producing Th(sup) (Th3-like). Our findings reconcile conflicting results by clearly demonstrating that suppression through naturally occurring CD25(+) Tregs is primary cell-contact-dependent but is subsequently followed by cell-contact-independent T cell inhibition mediated by second-generation Tr1- and Th3-like Th(sup) via the soluble factors IL-10 and TGF-beta.