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The functional and phenotypic properties of normal human CD3+CD5- T cells which have a higher frequency of cytotoxic cells than CD3+CD5+ T lymphocytes have been described. Using three- and four-colour immunofluorescence flow cytometric cell sorting, the CD3+CD5- and CD3+CD5+ populations were subdivided into αβ or γδ T cell receptor positive cells. The four subsets were examined for the in vitro cytotoxic activity and were also stimulated with mitogens in limiting-dilution assays to measure the frequencies of proliferating and interleukin-2 (IL-2) producing cells. CD+CD5-αβ+, CD3+CD5-γδ+ and CD3+CD5+γδ+ cells had lower frequencies of proliferating and IL-2-producing cells than did CD3+CD5+αβ+ cells. However, the cytotoxic activity of the different phenotypes was higher in the CD3+CD5- subsets, especially when these cells were γδ+. Expression of γδ or lack of expression of CD5 appeared to be associated with the acquisition of cytolytic potentials. CD8 was expressed on 20% of fresh CD3+γδ+ cells. Cultured γδ+ cells retained the expression of γδ, but quickly lost that of CD8 and with time modulated the expression of CD5. The expression of CD5 was found to be higher on sorted CD3+CD5+γδ- than on CD3+CD5-γδ+ cells. These observations indicate that γδ is preferentially expressed on CD5-negative or weakly positive T lymphocytes and that CD3+CD5-γδ+ cells appear to constitute a discrete small subset of mature T lymphocytes which are cytotoxic in nature. However, the exact immunological function of these cells and their place in T cell ontogeny are yet to be elucidated. [ABSTRACT FROM AUTHOR]

The functional and phenotypic properties of normal human CD3+CD5- T cells which have a higher frequency of cytotoxic cells than CD3+CD5+ T lymphocytes have been described. Using three- and four-colour immunofluorescence flow cytometric cell sorting, the CD3+CD5- and CD3+CD5+ populations were subdivided into alpha beta or gamma delta T cell receptor positive cells. The four subsets were examined for the in vitro cytotoxic activity and were also stimulated with mitogens in limiting-dilution assays to measure the frequencies of proliferating and interleukin-2 (IL-2) producing cells. CD3+CD5- alpha beta +, CD3+CD5- gamma delta + and CD3+CD5+ gamma delta + cells had lower frequencies of proliferating and IL-2-producing cells than did CD3+CD5+ alpha beta + cells. However, the cytotoxic activity of the different phenotypes was higher in the CD3+CD5- subsets, especially when these cells were gamma delta +. Expression of gamma delta or lack of expression of CD5 appeared to be associated with the acquisition of cytolytic potentials. CD8 was expressed on 20% of fresh CD3+ gamma delta + cells. Cultured gamma delta + cells retained the expression of gamma delta, but quickly lost that of CD8 and with time modulated the expression of CD5. The expression of CD5 was found to be higher on sorted CD3+CD5+ gamma delta - than on CD3+CD5+ gamma delta + cells. These observations indicate that gamma delta is preferentially expressed on CD5-negative or weakly positive T lymphocytes and that CD3+CD5- gamma delta + cells appear to constitute a discrete small subset of mature T lymphocytes which are cytotoxic in nature. However, the exact immunological function of these cells and their place in T cell ontogeny are yet to be elucidated.

In this report we describe the use of xantham gum as a biologically inert material for increasing the viscosity of a suspension of cells or particles during flow cytometric analysis and sorting. A 0.1% concentration of xantham gum in culture medium or saline will increase the viscosity approximately 9-fold. For suspensions of multicellular spheroids 100-400 microns in diameter the measured sedimentation velocity was approximately 9 times slower than that in medium alone. Thus, spheroids of 100 microns diameter remain in suspension in 0.1% xantham gum for 66 min, compared to 7.5 min in culture medium. This allows extended periods of sorting without stirring or agitating the sample suspension. The xantham gum solution is noncytotoxic for periods up to 8 h as measured by clonogenicity assay. Xantham gum has the added advantage that the viscosity is significantly reduced when the solution is subjected to shear stress, such as during flow. This technique should be applicable to extended sorting of suspensions of spheroids, plant cells, and other large particles, as well as for analyzing and sorting single cells for extended periods.

Phenotypes of lung free cells (FC) harvested from Fischer-344 rats by episodic lavage were characterized by flow cytometry. Parameters evaluated included electronic volume (EV), axial light loss (ALL), 90 degrees light scatter (LS), blue autofluorescence (BA), and green-yellow autofluorescence (G-YA). Three phenotypic populations, FC-A, FC-B, and FC-C were identified by their differing LS characteristics. FC-C represented 90% of the cells and were exclusively alveolar macrophages. Two subpopulations in FC-C, FC-CI and FC-CII, were further distinguished by their unique ALL features. The morphologic appearances of these subpopulations by light microscopy clearly differed in sorted preparations. Based on their patterns of autofluorescence, these FC-CI and FC-CII phenotypes were found to be composed of eight subpopulations. In FC populations harvested during further lavage episodes of the same lungs, the relative contributions of FC-CI to the FC-C subpopulation decreased as FC-CII correspondingly increased. This study demonstrates 1) that subpopulations of lavaged AM can be categorized according to their optical phenotypes by flow cytometry and 2) that the relative frequency of retrieval of some phenotypes depends on how exhaustively the lungs are lavaged. With regard to the latter, bronchoalveolar lavage does not randomly sample the underlying AM population in the alveolar compartment.