Alexander H. Enk

Infectious Tolerance Human CD25+ Regulatory T Cells Convey Suppressor Activity to Conventional CD4+ T Helper Cells

Regulatory CD4+CD25+ T cells (Treg) are mandatory for maintaining immunologic self-tolerance. We demonstrate that the cell-cell contact–mediated suppression of conventional CD4+ T cells by human CD25+ Treg cells is fixation resistant, independent from membrane-bound TGF-β but requires activation and protein synthesis of CD25+ Treg cells. Coactivation of CD25+ Treg cells with Treg cell–depleted CD4+ T cells results in anergized CD4+ T cells that in turn inhibit the activation of conventional, freshly isolated CD4+ T helper (Th) cells. This infectious suppressive activity, transferred from CD25+ Treg cells via cell contact, is cell contact–independent and partially mediated by soluble transforming growth factor (TGF)-β. The induction of suppressive properties in conventional CD4+ Th cells represents a mechanism underlying the phenomenon of infectious tolerance. This explains previously published conflicting data on the role of TGF-β in CD25+ Treg cell–induced immunosuppression.

A comparison of two types of dendritic cell as adjuvants for the induction of melanoma-specific T-cell responses in humans following intranodal injection

Dendritic cells (DCs) elicit potent anti‐tumoral T‐cell responses in vitro and in vivo. However, different types of DC have yet to be compared for their capacity to induce anti‐tumor responses in vivo at different developmental stages. Herein, we correlated the efficiencies of different types of monocyte‐derived DC as vaccines on the resulting anti‐tumor immune responses in vivo. Immature and mature DCs were separately pulsed with a peptide derived from tyrosinase, MelanA/MART‐1 or MAGE‐1 and a recall antigen. Both DC populations were injected every 2 weeks in different lymph nodes of the same patient. Immune responses were monitored before, during and after vaccination. Mature DCs induced increased recall antigen‐specific CD4+ T‐cell responses in 7/8 patients, while immature DCs did so in only 3/8. Expansion of peptide‐specific IFN‐γ–producing CD8+ T cells was observed in 5/7 patients vaccinated with mature DCs but in only 1/7 using immature DCs. However, these functional data did not correlate with the tetramer staining. Herein, immature DCs also showed expansion of peptide‐specific T cells. In 2/4 patients vaccinated with mature DCs, we observed induction of peptide‐specific cytotoxic T cells, as monitored by chromium‐release assays, whereas immature DCs failed to induce peptide‐specific cytotoxic T cells in the same patients. Instead, FCS‐cultured immature DCs induced FCS‐specific IgE responses in 1 patient. Our data demonstrate that this novel vaccination protocol is an efficient approach to compare different immunization strategies within the same patient. Thus, our data define FCS‐free cultured mature DCs as superior inducers of T‐cell responses in melanoma patients.

Dendritic cells as mediators of tumor-induced tolerance in metastatic melanoma

Escape from immune surveillance is critical for tumor progression in metastatic melanoma. We assessed the function of melanoma‐derived dendritic cells (DCs) in patients presenting simultaneously with responding (rM) or progressing (pM) melanoma metastases. These rare coincidences allowed us to compare syngeneically the function of tumor DCs. CD83+ DCs were purified freshly from large responding (rDCs) or progressing (pDCs) metastases following chemo‐immunotherapy. rDCs were 5 times more potent inducers of allogeneic T‐cell proliferation than the pDCs that were used as control. Phenotypic analysis showed a marked depression of CD86 expression on pDCs. Culture supernatants from pM showed production of Th2‐type cytokines [interleukin‐10 (IL‐10)], whereas a Th1 pattern [IL‐2], interferon‐γ (IFN‐γ), IL‐12) predominated in rM. The IL‐10 detected in progressing metastases was directly derived from melanoma cells. Culture supernatants from metastases applied to DC‐supported allo‐MLR assays suppressed T‐cell responses by 50–75% in the case of pM, but not rM. Finally, in a co‐stimulation‐dependent anti‐CD3 tolerance assay, pDCs (but not rDCs) induced anergy in syngeneic CD4+ T cells. Anergy could be overcome by addition of IL‐12 or IL‐2. Our results show that melanoma‐derived factors convert DC‐antigen presenting cell function to tolerance induction against tumor tissue, changing tumor DCs to “silencers” of anti‐tumoral immune responses. Int. J. Cancer 73:309–316, 1997.

Immature, but not inactive: the tolerogenic function of immature dendritic cells

The induction of antigen‐specific T cell tolerance and its maintenance in the periphery is critical for the prevention of autoimmunity. Recent evidence shows that dendritic cells (DC) not only initiate T cell responses, but are also involved in silencing of T cell immune responses. The functional activities of DC are mainly dependent on their state of activation and differentiation, that is, terminally differentiated mature DC can efficiently induce the development of T effector cells, whereas immature DC are involved in maintenance of peripheral tolerance. The means by which immature DC maintain peripheral tolerance are not entirely clear, however, their functions include the induction of anergic T cells, T cells with regulatory properties as well as the generation of T cells that secrete immunomodulatory cytokines. This review summarizes the current knowledge about the immunoregulatory role of immature DC that might act as guardians for the induction and maintenance of T cell tolerance in the periphery.

Production of functional IL‐18 by different subtypes of murine and human dendritic cells (DC): DC‐derived IL‐18 enhances IL‐12‐dependent Th1 development

IL‐18 is a recently described cytokine that shares biological activities with IL‐12 in driving the development of Th1‐type T cells. As dendritic cells (DC) are very potent inducers of T cell proliferation and differentiation we wondered whether they utilize IL‐18 as a factor driving Th1 development. We demonstrate by Northern blot and reverse transcription‐PCR that various subtypes of human and murine DC as well as the DC‐line XS contain IL‐18 mRNA. When supernatants of either enriched Langerhans cells (LC) or bone marrow‐derived DC were analyzed for production of IL‐18 protein, IL‐18 production was detected in an IL‐18‐specific ELISA. To assess whether the IL‐18 protein released by DC is functional, we performed a sensitive bioassay using the IL‐18‐dependent stimulation of concanavalin A‐stimulated T cells. Both, supernatants from bone marrow‐derived DC and enriched LC induced IFN‐γ production in the T cells. This production was partially inhibitable by addition of anti‐IL‐18 antiserum. In a TCR‐transgenic mouse system we further demonstrate that DC‐derived IL‐18 potentiates IL‐12‐dependent Th1 development. Using DC derived from IL‐12 knockout animals, we show that DC‐derived IL‐18 by itself is not capable of inducing Th1 cell differentiation. Together the data demonstrate that subtypes of DC are able to release functional IL‐18 that is able to induce IFN‐γ production and Th1 differentiation in primed T cells.

Mage-3 and Influenza-Matrix Peptide-Specific Cytotoxic T Cells Are Inducible in Terminal Stage HLA-A2.1+ Melanoma Patients by Mature Monocyte-Derived Dendritic Cells

Dendritic cell (DC) vaccination, albeit still in an early stage, is a promising strategy to induce immunity to cancer. We explored whether DC can expand Ag-specific CD8+ T cells even in far-advanced stage IV melanoma patients. We found that three to five biweekly vaccinations of mature, monocyte-derived DC (three vaccinations of 6 × 106 s.c. followed by two i.v. ones of 6 and 12 × 106, respectively) pulsed with Mage-3A2.1 tumor and influenza matrix A2.1-positive control peptides as well as the recall Ag tetanus toxoid (in three of eight patients) generated in all eight patients Ag-specific effector CD8+ T cells that were detectable in blood directly ex vivo. This is the first time that active, melanoma peptide-specific, IFN-γ-producing, effector CD8+ T cells have been reliably observed in patients vaccinated with melanoma Ags. Therefore, our DC vaccination strategy performs an adjuvant role and encourages further optimization of this new immunization approach.

Human CD25+ regulatory T cells: two subsets defined by the integrins α4β7 or α4β1 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(Thsup), which inhibit T cell activation via soluble mediators. Here, we describe two distinct subsets of human Tregs, characterized by expression of either the α4β7 integrin or the α4β1 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: α4β7+Tregs induce IL‐10‐producing Thsup (Tr1‐like), whereas α4β1+ Tregs induce TGF‐β‐producing Thsup (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 Thsup via the soluble factors IL‐10 and TGF‐β.

Different Efficiency of Heat Shock Proteins (HSP) to Activate Human Monocytes and Dendritic Cells: Superiority of HSP60

One essential immunoregulatory function of heat shock protein (HSP) is activation of the innate immune system. We investigated the activation of human monocytes and monocyte-derived dendritic cells (DC) by recombinant human HSP60, human inducible HSP72, and preparations of human gp96 and HSP70 under stringent conditions, in the absence of serum and with highly purified monocytes. HSP60 induced human DC maturation and activated human DC to secrete proinflammatory cytokines. HSP72 induced DC maturation to a lesser extent, but activated human monocytes and immature DC as efficiently as HSP60 to release proinflammatory cytokines. The independence of the effects of HSP60 and HSP72 from endotoxin or another copurifying bacterial component was shown by the resistance of these effects to polymyxin B, their sensitivity to heat treatment, the inactivity of endotoxin controls at concentrations up to 100-fold above the endotoxin contents of the HSP, and the inactivity of a recombinant control protein. Preparations of HSP70, which consisted mainly of the constitutively expressed HSP73, induced only marginal cytokine release from monocytes. The gp96 preparations did not have significant effects on human monocytes and monocyte-derived DC, indicating that these human APC populations were not susceptible to gp96 signaling under the stringent conditions applied in this study. The biological activities of gp96 and HSP70 preparations were confirmed by their peptide binding activity. These findings show that HSP can differ considerably in the capacity to activate monocyte-derived APC under certain conditions and underline the potential of HSP60 and HSP72 as activation signals for the innate immune system.

Identification and induction of human keratinocyte-derived IL-12.

Interleukin 12 is a heterodimeric molecule that serves as a potent co-stimulator enhancing the development of Th1 cells. As one of the classical Th1 cell-mediated responses is contact sensitivity in skin, we wondered whether IL-12 might be produced by epidermal cells and serve as a mediator of this immune response. Using a sensitive, quantitative PCR technique we demonstrate that p35 chain mRNA of IL-12 is produced constitutively by human epidermal cells, whereas p40 chain mRNA can only be detected in epidermis treated with contact allergen, but not epidermis exposed to irritants or tolerogens. Time course studies showed a dramatic induction of IL-12 p40 mRNA 4 h after in vivo allergen treatment reaching peak strength after 6 h. In cell depletion assays we show that epidermal keratinocytes are the major source of this cytokine in the epidermis. This was further supported by analysis of mRNA derived from the human keratinocyte cell line HaCat expressing IL-12 p35 and p40 mRNA upon stimulation. The presence of bioactive IL-12 in supernatants derived from allergen-stimulated epidermal cells was demonstrated by IL-12-specific bioassay. Additional evidence for the functional importance of IL-12 in primary immune reactions in skin was obtained in allogeneic proliferation assays using human haptenated epidermal cells containing Langerhans cells as APC and allogeneic CD4+ T cells as responders. Anti-IL-12 mAb inhibited the proliferation of T cells by approximately 50%. In aggregate our data demonstrate that nonlymphoid keratinocytes are capable of producing functional IL-12 and provide evidence for the functional significance of IL-12 in primary immune responses in skin.

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.