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Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry
Published on: April 19, 2017
A new method for measuring clustering in suspension between accessory cells and T lymphocytes
V A Gant1, Z Shakoor, A S Hamblin
1Department of Immunology, UMDS, London, UK.
Insights
Researchers developed a new method to measure cell clusters between monocytes and T lymphocytes. This technique helps understand immune responses to antigens and mitogens, revealing distinct activation pathways.
Area of Science:
- Immunology
- Cell Biology
- Molecular Medicine
Background:
- Understanding cell-cell interactions, specifically cluster formation between antigen-presenting cells (monocytes) and T lymphocytes, is crucial in immunology.
- The molecular basis and clinical significance of these clusters are not fully elucidated, hindering advancements in immune response studies.
Purpose of the Study:
- To develop and validate a novel, reproducible technique for quantifying cluster formation between human monocytes and T lymphocytes in suspension.
- To apply this method to assess the impact of recall antigens and mitogens on monocyte-T lymphocyte clustering.
Main Methods:
- Purified human blood monocytes and autologous T lymphocytes were co-cultured in suspension after labeling with distinct carbocyanine dyes.
- Cluster formation was measured at 24 hours using ultraviolet microscopy and flow cytometry.
- Control experiments confirmed dye non-toxicity, stability, and lack of leakage.
Main Results:
- A strong correlation was observed between microscopic and flow cytometric measurements of monocyte:T lymphocyte clustering (p < 0.001).
- Recall antigens significantly increased clustering compared to baseline levels (p < 0.001), independent of baseline clustering.
- Mitogen (PHA)-induced clustering correlated with baseline levels (p = 0.02), suggesting different underlying mechanisms.
Conclusions:
- The developed technique provides a reliable method for measuring cell-cell associations in suspension over extended culture periods.
- The findings suggest distinct mechanisms govern antigen- and mitogen-induced monocyte-T lymphocyte clustering.
- This method is valuable for studying agents that modulate intercellular adhesion and immune responses.
Abstract:
Specifying the molecular basis and clinical significance of cluster formation between antigen-presenting cells and T lymphocytes will be important in many areas of immunology. In this paper we describe a novel and reproducible technique for measuring cluster formation in suspension between purified human blood monocytes and purified autologous T lymphocytes, and its application to determining the effects of recall antigens and mitogen. Blood monocytes and T lymphocytes from eight normal subjects were separately prelabelled with two different carbocyanine dyes prior to co-culture in suspension with or without antigen (PPD, SKSD) or mitogen (PHA). At 24 h the co-cultures were examined for cluster formation by ultraviolet microscopy and flow cytometry. Control experiments showed that the carbocyanine dyes were non-toxic in vitro, that cell labelling was stable for culture periods up to 120 h, and that the two dyes did not leak from cell to cell. By this technique we measured the proportion of monocytes clustering one or more T lymphocytes in the presence and absence of recall antigen or PHA. There was a close correlation between visual and flow cytometric measurement of monocyte: T lymphocyte clustering (p < 0.001) as well as a close relationship between the ability of the two recall antigens to increase the extent of clustering above baseline (p < 0.001). Antigen-increased cluster formation did not correlate with baseline clustering, unlike PHA-increased clustering, which was related to baseline levels (p = 0.02), suggesting the operation of distinct mechanisms. The method is applicable to measuring cell-cell associations in suspension during extended periods of culture, as well as for the study of agents which might modify intercellular adhesion processes.
