Jürgen Knop

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.

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.

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.

Decreased release of histamine and sulfidoleukotrienes by human peripheral blood leukocytes after wasp venom immunotherapy is partially due to induction of IL-10 and IFN-γ production of T cells

BACKGROUND Recent studies provide evidence that venom immunotherapy (VIT) alters the pattern of cytokine production by inducing an allergen-specific T-cell shift in cytokine expression from TH2 (IL-4, IL-5) to TH1 (IFN-gamma) cytokines and also inducing the production of IL-10. OBJECTIVE This study was carried out to analyze whether these changes in cytokine production of T cells already observed 1 week after the initiation of VIT in subjects with wasp venom allergy also influence the reactivity of effector cells, such as mast cells and basophils. METHODS All subjects included in this study had a history of severe systemic allergic reactions to wasp stings and positive skin test responses with venom and venom-specific IgE in the sera. Peripheral blood leukocytes were isolated before and after the initiation of VIT (rush therapy reaching a maintenance dose of 100 microg venom injected subcutaneously within 1 week) and preincubated with or without addition of IL-10, IFN-gamma, IL-10 + IFN-gamma, anti-IL-10, or anti-IFN-gamma. After stimulation with wasp venom, histamine and sulfidoleukotriene release were assessed by ELISA and compared with spontaneous release and total histamine content. RESULTS After the induction of VIT, venom-induced absolute and relative histamine and sulfidoleukotriene release were reduced. This was at least partially due to the induction of IFN-gamma and IL-10 production, because (1) neutralization of IL-10 and IFN-gamma by mAbs partially restored the release after the initiation of VIT and (2) the addition of exogenous IFN-gamma and IL-10 caused a statistically significant diminution of the venom-induced histamine and sulfidoleukotriene release before VIT. Depletion of CD2(+) T cells also restored the releasability after VIT. CONCLUSION These data indicate that T cells (producing IL-10 and IFN-gamma after VIT) play a key role for the inhibition of histamine and sulfidoleukotriene release of effector cells.

Interleukin 1α Promotes Th1 Differentiation and Inhibits Disease Progression in Leishmania major–susceptible BALB/c Mice

Protective immunity against pathogens such as Leishmania major is mediated by interleukin (IL)-12–dependent Th1-immunity. We have shown previously that skin-dendritic cells (DCs) from both resistant C57BL/6 and susceptible BALB/c mice release IL-12 when infected with L. major, and infected BALB/c DCs effectively vaccinate against leishmaniasis. To determine if cytokines other than IL-12 might influence disease outcome, we surveyed DCs from both strains for production of a variety of cytokines. Skin-DCs produced significantly less IL-1α in response to lipopolysaccharide/interferon γ or L. major when expanded from BALB/c as compared with C57BL/6 mice. In addition, IL-1α mRNA accumulation in lymph nodes of L. major–infected BALB/c mice was ∼3-fold lower than that in C57BL/6 mice. Local injections of IL-1α during the first 3 d after infection led to dramatic, persistent reductions in lesion sizes. In L. major–infected BALB/c mice, IL-1α administration resulted in increased Th1- and strikingly decreased Th2-cytokine production. IL-1α and IL-12 treatments were similarly effective, and IL-1α efficacy was strictly IL-12 dependent. These data indicate that transient local administration of IL-1α acts in conjunction with IL-12 to influence Th-development in cutaneous leishmaniasis and prevents disease progression in susceptible BALB/c mice, perhaps by enhancing DC-induced Th1-education. Differential production of IL-1 by C57BL/6 and BALB/c mice may provide a partial explanation for the disparate outcomes of infection in these mouse strains.

Treatment of pemphigus vulgaris with mycophenolate mofetil

different pattern of diffusion changes in a 51-year-old woman with chronic epilepsy and recurrent episodes of focal status epilepticus, for whom no aetiology could be established. Status consisted of clonic jerking of the right leg, which continued for 22 days and was followed by transient paresis. DWI during status showed decreased diffusion in the motor cortex of the right leg (relative decrease in ADC of 27%, see figure). Surprisingly, the diffusion was increased in the subcortical white matter (relative increase in ADC of 31%). On the T2-weighted image (not shown), both cortex and subcortical white matter of the corresponding region returned a high signal similar to previously reported cases. 4 After cessation of the status, during transient paresis, repeat DWI and T2-weighted imaging showed resolution of these abnormalities. Resolution of diffusion changes preceded functional recovery. The pattern of diffusion changes observed by us differed from the changes observed so far in brain ischaemia and experimental status, since the decrease in cortical diffusion was associated with increased diffusion in the subcortical white matter. A possible explanation for the pattern of diffusion changes is water shifts associated with protracted epileptic activity. During epileptic activity, the extracellular space shrinks due to a water flux into cells at the area of maximum neuronal activity (in the cortex), 5 which would

Skin mast cells control T cell-dependent host defense in Leishmania major infections

Mast cells (MCs) initiate protective im‐ munity against bacteria. Here we demonstrate that MCs also contribute to the control of parasitic skin infections by Leishmania major. L. major‐infected MC‐defi‐cient KitW/KitW‐v mice developed markedly larger skin lesions than did normal Kit+/+ mice (>2‐fold), and cutaneous reconstitution with MCs resulted in normalization of lesion development. KitW/KitW‐v lesions contained significantly more parasites, and infections resulted in enhanced spreading of parasites to the spleens as compared to controls. In addition, recruitment of proinflammatory neutrophils, macrophages, and dendritic cells (DCs) in infected mice was MC dependent. In the absence of MCs, reduced numbers of lesional DCs were associated with decreased production of Th1‐promoting interleukin (IL)‐12. Antigen‐specific T cell priming was delayed in KitW/KitW‐v mice and cytokine responses were skewed towards Th2. Notably, local skin MC reconstitution at sites of infection was sufficient for the induction of systemic protection. Thus, MC‐mediated control of L. major infections is not limited to the induction of local inflammation. Instead, MCs contribute significantly to local DC recruitment, which mediates protective immunity. These findings extend the view of MCs as salient sentinels of innate immunity to complex host defense reactions against intracellular pathogens.—Maurer, M., Lopez Kostka, S., Siebenhaar, F., Moelle, K., Metz, M., Knop, J., von Stebut, E. Skin mast cells control T cell‐dependent host defense in Leishmania major infections. FASEB J. 20, 2460–2467 (2006)