Maike Blessenohl

Human peripheral γδ T cells possess regulatory potential

Deficiency in γδ T cells aggravates colitis in animal models suggesting that γδ T cells have regulatory properties. Therefore, proliferation, suppression and cytokine secretion of human γδ T cells were determined in vitro. Human peripheral γδ T cells were isolated from the whole blood of healthy donors by magnetic antibody cell sorting technology. The proliferation after CD3/CD28 stimulation was measured by 3[H]thymidine incorporation. Interferon‐γ (IFN‐γ), interleukin‐2 (IL‐2), transforming growth factor‐β (TGF‐β) and IL‐10 concentrations were measured by enzyme‐linked immunosorbent assay; TGF‐β messenger RNA was also measured by reverse transcription–polymerase chain reaction. The expression of latency associated peptide (LAP), a TGF‐β complex component, intracellular cytokine content and T helper cell proliferation were measured by flow cytometry. Human γδ T cells showed poor proliferation upon CD3/CD28 stimulation and suppressed T helper cell growth stronger than CD4+ CD25+ T cells, although γδ T cells were FOXP3 negative. They secreted little IL‐2 but high concentrations of IFN‐γ, IL‐10 and TGF‐β. When looking at LAP expression the Vδ1 subset was found to be the main TGF‐β producer compared to Vδ2 T cells. Taken together, peripheral γδ T cells have in vitro a more potent regulatory potential than CD4+ CD25+ cells regarding T helper cell suppression. This is most likely the result of strong TGF‐β secretion, particularly by the Vδ1 subset.

Human peripheral gammadelta T cells possess regulatory potential.

Deficiency in γδ T cells aggravates colitis in animal models suggesting that γδ T cells have regulatory properties. Therefore, proliferation, suppression and cytokine secretion of human γδ T cells were determined in vitro. Human peripheral γδ T cells were isolated from the whole blood of healthy donors by magnetic antibody cell sorting technology. The proliferation after CD3/CD28 stimulation was measured by 3[H]thymidine incorporation. Interferon‐γ (IFN‐γ), interleukin‐2 (IL‐2), transforming growth factor‐β (TGF‐β) and IL‐10 concentrations were measured by enzyme‐linked immunosorbent assay; TGF‐β messenger RNA was also measured by reverse transcription–polymerase chain reaction. The expression of latency associated peptide (LAP), a TGF‐β complex component, intracellular cytokine content and T helper cell proliferation were measured by flow cytometry. Human γδ T cells showed poor proliferation upon CD3/CD28 stimulation and suppressed T helper cell growth stronger than CD4+ CD25+ T cells, although γδ T cells were FOXP3 negative. They secreted little IL‐2 but high concentrations of IFN‐γ, IL‐10 and TGF‐β. When looking at LAP expression the Vδ1 subset was found to be the main TGF‐β producer compared to Vδ2 T cells. Taken together, peripheral γδ T cells have in vitro a more potent regulatory potential than CD4+ CD25+ cells regarding T helper cell suppression. This is most likely the result of strong TGF‐β secretion, particularly by the Vδ1 subset.

T Cell Zones of Lymphoid Organs Constitutively Express Th1 Cytokine mRNA: Specific Changes during the Early Phase of an Immune Response

The cytokine milieu of the T cell zones in lymphoid organs is involved in the activation of naive T cells. Quantitative data regarding the local expression of cytokines are lacking. Therefore, the expression of Th1 (IL-2, IL-12p40, IFN-γ), Th2 (IL-4, IL-10), as well as TGFβ1 and IL-15 mRNA was studied after laser microdissection in the steady state and during an immune response in rats. Our results show that Th1 cytokines are preferentially found in lymphoid tissues and in the T cell zones, whereas Th2 cytokines are expressed throughout the organs and especially in the B cell zones. After injection of sheep RBC, IL-2 and IFN-γ mRNA are significantly increased in the T cell zone only, a change not seen by analyzing the whole spleen. Studying the spatial and temporal expression of genes will reveal new insights into the regulation of immune responses.

In vivo spectral imaging of different cell types in the small intestine by two-photon excited autofluorescence

Spectrally resolved two-photon excited autofluorescence imaging is used to distinguish different cell types and functional areas during dynamic processes in the living gut. Excitation and emission spectra of mucosal tissue and tissue components are correlated to spectra of endogenous chromophores. We show that selective excitation with only two different wavelengths within the tuning range of a Ti:sapphire femtosecond laser system yields excellent discrimination between enterocytes, antigen presenting cells and lysosomes based on the excitation and emission properties of their autofluorescence. The method is employed for time-lapse microscopy over up to 8 h. Changes of the spectral signature with the onset of photodamage are demonstrated, and their origin is discussed.

Complex morphology and functional dynamics of vital murine intestinal mucosa revealed by autofluorescence 2-photon microscopy

The mucosa of the gastrointestinal tract is a dynamic tissue composed of numerous cell types with complex cellular functions. Study of the vital intestinal mucosa has been hampered by lack of suitable model systems. We here present a novel animal model that enables highly resolved three-dimensional imaging of the vital murine intestine in anaesthetized mice. Using intravital autofluorescence 2-photon (A2P) microscopy we studied the choreographed interactions of enterocytes, goblet cells, enteroendocrine cells and brush cells with other cellular constituents of the small intestinal mucosa over several hours at a subcellular resolution and in three dimensions. Vigorously moving lymphoid cells and their interaction with constituent parts of the lamina propria were examined and quantitatively analyzed. Nuclear and lectin staining permitted simultaneous characterization of autofluorescence and admitted dyes and yielded additional spectral information that is crucial to the interpretation of the complex intestinal mucosa. This novel intravital approach provides detailed insights into the physiology of the small intestine and especially opens a new window for investigating cellular dynamics under nearly physiological conditions.

TNF Receptor-1 Is Required for the Formation of Splenic Compartments during Adult, but Not Embryonic Life

Lymphotoxin β-receptor (LTβR) and TNF receptor-1 (TNFR1) are important for the development of secondary lymphoid organs during embryonic life. The significance of LTβR and TNFR1 for the formation of lymphoid tissue during adult life is not well understood. Immunohistochemistry, morphometry, flow cytometry, and laser microdissection were used to compare wild-type, LTβR−/−, TNFR1−/− spleens with splenic tissue that has been newly formed 8 wk after avascular implantation into adult mice. During ontogeny, LTβR is sufficient to induce formation of the marginal zone, similar-sized T and B cell zones, and a mixed T/B cell zone that completely surrounded the T cell zone. Strikingly, in adult mice, the formation of splenic compartments required both LTβR and TNFR1 expression, demonstrating that the molecular requirements for lymphoid tissue formation are different during embryonic and adult life. Thus, interfering with the TNFR1 pathway offers the possibility to selectively block the formation of ectopic lymphoid tissue and at the same time to spare secondary lymphoid organs such as spleen and lymph nodes. This opens a new perspective for the treatment of autoimmune and inflammatory diseases.

Analyzing the Migration of Labeled T Cells In Vivo: An Essential Approach with Challenging Features

T cells are involved in the pathogenesis of many diseases. To exert a pathological effect, T cells enter the tissues. We show that the determination of their entry site requires isolation of the respective T cell population, injection into genetically un-manipulated animals, and identification of the cells in vivo at various time points after injection. We indicate variables influencing in vivo migration experiments artificially, and outline how resulting problems can be either avoided or taken into account. Reviewing experiments performed according to the outlined criteria reveals two types of migration patterns for T cell subsets in vivo: 1) Naïve and memory T cells enter lymphoid and non-lymphoid organs in comparable numbers, but selectively accumulate in lymphoid tissues over time, 2) Effector T cells, too, enter lymphoid and non-lymphoid organs in comparable numbers. However, most of them die within 24 hours. Depending on the presence of cytokines, chemokines and extracellular matrix compounds they are able to survive, thereby preferentially accumulating in their target tissues. This information might help to understand the role of migration in the pathogenesis of T cell mediated diseases.

Dendritic cell subsets in lymph nodes are characterized by the specific draining area and influence the phenotype and fate of primed T cells

Dendritic cells (DC) are important in differential T‐cell priming. Little is known about the local priming by DC in the microenvironment of different lymph nodes and about the fate of the imprinted T cells. Therefore, freshly isolated rat DC from mesenteric lymph nodes (mLN) and axillary lymph nodes (axLN) were phenotyped and cultured with blood T cells in the presence of the superantigen Mycoplasma arthritidis mitogen (MAM). The phenotype, proliferation and apoptosis of the primed T cells were analysed. Our data show that a common DC population exists in both mLN and axLN. In addition, region‐specific DC with an organotypical marker expression imprinted by the drained area were found. Coculture of T cells with DC from mLN or axLN resulted in a distinct shift in the CD4 and CD8 expression of T cells and their phenotype. Furthermore, when these differentially primed mLN and axLN T cells were injected into recipients, mLN‐primed T cells survived longer in other lymphoid organs. The results show that the region‐specific DC have a unique phenotype and an impact on the ratio of CD4 : CD8 T cells during an immune response in vivo.

Human peripheral γδ T cells possess regulatory potential: Human γδ T cells have regulatory potential

Deficiency in γδ T cells aggravates colitis in animal models suggesting that γδ T cells have regulatory properties. Therefore, proliferation, suppression and cytokine secretion of human γδ T cells were determined in vitro. Human peripheral γδ T cells were isolated from the whole blood of healthy donors by magnetic antibody cell sorting technology. The proliferation after CD3/CD28 stimulation was measured by 3[H]thymidine incorporation. Interferon‐γ (IFN‐γ), interleukin‐2 (IL‐2), transforming growth factor‐β (TGF‐β) and IL‐10 concentrations were measured by enzyme‐linked immunosorbent assay; TGF‐β messenger RNA was also measured by reverse transcription–polymerase chain reaction. The expression of latency associated peptide (LAP), a TGF‐β complex component, intracellular cytokine content and T helper cell proliferation were measured by flow cytometry. Human γδ T cells showed poor proliferation upon CD3/CD28 stimulation and suppressed T helper cell growth stronger than CD4+ CD25+ T cells, although γδ T cells were FOXP3 negative. They secreted little IL‐2 but high concentrations of IFN‐γ, IL‐10 and TGF‐β. When looking at LAP expression the Vδ1 subset was found to be the main TGF‐β producer compared to Vδ2 T cells. Taken together, peripheral γδ T cells have in vitro a more potent regulatory potential than CD4+ CD25+ cells regarding T helper cell suppression. This is most likely the result of strong TGF‐β secretion, particularly by the Vδ1 subset.