SummaryDeficiency in cd T cells aggravates colitis in animal models suggesting that cd T cells have regulatory properties. Therefore, proliferation, suppression and cytokine secretion of human cd T cells were determined in vitro. Human peripheral cd 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-c (IFN-c), interleukin-2 (IL-2), transforming growth factor-b (TGF-b) and IL-10 concentrations were measured by enzymelinked immunosorbent assay; TGF-b messenger RNA was also measured by reverse transcription-polymerase chain reaction. The expression of latency associated peptide (LAP), a TGF-b complex component, intracellular cytokine content and T helper cell proliferation were measured by flow cytometry. Human cd T cells showed poor proliferation upon CD3/ CD28 stimulation and suppressed T helper cell growth stronger than CD4 + CD25 + T cells, although cd T cells were FOXP3 negative. They secreted little IL-2 but high concentrations of IFN-c, IL-10 and TGF-b. When looking at LAP expression the Vd1 subset was found to be the main TGF-b producer compared to Vd2 T cells. Taken together, peripheral cd 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-b secretion, particularly by the Vd1 subset.

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.

The discovery that bovine peripheral lymphocytes are sensitive to Stx1 identified a possible mechanism for the persistence of infections with Shiga toxin (Stx)-producing Escherichia coli (STEC) in the bovine reservoir host. If intraepithelial lymphocytes (IEL) are also sensitive to Stx1, the idea that Stx1 affects inflammation in the bovine intestine is highly attractive. To prove this hypothesis, ileal IEL (iIEL) were prepared from adult cattle, characterized by flow cytometry, and subjected to functional assays in the presence and absence of purified Stx1. We found that 14.9% of all iIEL expressed Gb 3 /CD77, the Stx1 receptor on bovine lymphocytes, and 7.9% were able to bind the recombinant B subunit of Stx1. The majority of Gb 3 /CD77 ؉ cells were activated

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.

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00418-011-0905-0) contains supplementary material, which is available to authorized users.