Ag-specific CD4+ T cells orchestrating adaptive immune responses are crucial for the development of protective immunity, but also mediate immunopathologies. To date, technical limitations often prevented their direct analysis. In this study, we report a sensitive flow cytometric assay based on magnetic pre-enrichment of CD154+ T cells to visualize rare Ag-reactive naive and memory Th cells directly from human peripheral blood. The detection limit of ∼1 cell within 105–106 permitted the direct enumeration and characterization of auto-, tumor-, or neo-Ag–reactive T cells within the naive and even memory CD4+ T cell repertoire of healthy donors. Furthermore, the analysis of high target cell numbers after pre-enrichment of rare Ag-specific T cells from large blood samples dramatically improved the identification of small subpopulations. As exemplified in this work, the dissection of the Ag-specific memory responses into small cytokine-producing subsets revealed great heterogeneity between pathogens, but also pathogen-related microsignatures refining Th cell subset classification. The possibility to directly analyze CD4+ T cells reactive against basically any Ag of interest at high resolution within the naive and memory repertoire will open up new avenues to investigate CD4+ T cell–mediated immune reactions and their use for clinical diagnostics.
We investigated the in vivo effect of hyperthermia on the expression of heat shock proteins and MRI changes in three tumor cell lines. Three tumor cell lines (SCCVII, NIH3T3, M21) were transfected with a plasmid containing the heat shock protein 70 gene (hsp70) promoter fragment and the luciferase reporter gene, and injected into mice. Tumors of 1100 mm³ in size were exposed to five different temperatures (38, 40, 42, 44 and 46 °C) in a water bath. Bioluminescence and MRI were performed at set time intervals. The MRI scan protocol was as follows: T₁-weighted spin echo ± contrast medium, T₂-weighted fast spin echo, dynamic contrast-enhanced MRI, diffusion-weighted stimulated echo acquisition mode sequence, T₂ time obtained on a 1.5T General Electric MRI scanner. Immunoblotting was also performed. hsp70 transcription was strongly induced at 42 and 44 °C, reaching values as high as 8531.5 ± 432.1-fold above baseline in NIH3T3 tumors. At these temperatures, significant increases in the uptake of contrast medium, slope of initial enhancement, Ak(ep) values and apparent diffusion coefficient (ADC) were observed in the 8-h scan of the NIH3T3 cell line. In SCCVII tumors, ADC increased by about 23% (p = 0.010) in the scans performed at 8, 24, 48 and 96 h. At 46 °C, luciferase activity was reduced significantly in the three cell lines. In all tumor types, a significant increase in ADC was observed, which was highest in SCCVII tumors (33.8%; p < 0.01). In accordance with the bioluminescence results, significant Hsp70 protein production was shown by immunoblot analysis. The best correlation coefficient between luciferase activity and immunoblotting results was found for M21 tumors (r = 0.93, p < 0.0001). Different tissue types display distinct patterns of hsp70 transcription. MRI can be used, in combination with optical imaging, to provide information on hsp70 transcription and protein production. The major finding of the present study was that heat-related biochemical changes in tumor tissue can be determined by MRI.
CD4+ T cells play a central role in the immune defense against pathogens or tumors but also in immunopathology, such as autoimmunity or allergy. However, due to technical limitations, comprehensive information about the frequency and phenotype of antigen-specific CD4+ T cells against many disease-relevant antigens is missing. In particular the nature of the human naïve repertoire is elusive. We have developed a new, sensitive detection system for antigen-specific T helper cells, based on a combination of magnetic pre-enrichment and multi-parametric flow cytometric analysis of CD154 (CD40L)-expressing CD4+ T cells upon in vitro activation with antigens. The high sensitivity of 1 cell out of 105 to 106 enables the direct enumeration of memory and naïve CD4+ T cells reactive against basically any recall or primary antigen. Importantly, our technology allowed us to rapidly analyze sufficient numbers of target cells to discriminate even small functional or phenotypic subpopulations within the total antigen-specific T cell pool, undetectable by conventional methods. We used this assay for a first characterization of tumor- and auto-antigen reactive CD4+ T cells from healthy subjects as well as for the in depth characterization of rare pathogen specific T cells. The possibility to visualize T cells reactive against any antigen of interest even in the naïve repertoire will open up new possibilities to advance human T cell research and to improve clinical diagnostics and prognosis.
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