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Phenotypic and functional separation of memory and effector human CD8+ T cells.

Hamann D, Baars PA, Rep MH, Hooibrink B, Kerkhof-Garde SR, Klein MR, van Lier RA - J. Exp. Med. (1997)

Bottom Line: These T cells do not secrete IL-2 or -4 but can produce IFN-gamma and TNF-alpha.Interestingly, CD8+CD45RA+CD27- cells parallel effector CTLs, as they abundantly express Fas-ligand mRNA, contain perforin and granzyme B, and have high cytolytic activity without in vitro prestimulation.Based on both phenotypic and functional properties, we conclude that memory- and effector-type T cells can be separated as distinct entities within the human CD8+ T cell subset.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Viro-Immunology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, University of Amsterdam.

ABSTRACT
Human CD8+ memory- and effector-type T cells are poorly defined. We show here that, next to a naive compartment, two discrete primed subpopulations can be found within the circulating human CD8+ T cell subset. First, CD45RA-CD45R0(+) cells are reminiscent of memory-type T cells in that they express elevated levels of CD95 (Fas) and the integrin family members CD11a, CD18, CD29, CD49d, and CD49e, compared to naive CD8+ T cells, and are able to secrete not only interleukin (IL) 2 but also interferon gamma, tumor necrosis factor alpha, and IL-4. This subset does not exert cytolytic activity without prior in vitro stimulation but does contain virus-specific cytotoxic T lymphocyte (CTL) precursors. A second primed population is characterized by CD45RA expression with concomitant absence of expression of the costimulatory molecules CD27 and CD28. The CD8+CD45RA+CD27- population contains T cells expressing high levels of CD11a, CD11b, CD18, and CD49d, whereas CD62L (L-selectin) is not expressed. These T cells do not secrete IL-2 or -4 but can produce IFN-gamma and TNF-alpha. In accordance with this finding, cells contained within this subpopulation depend for proliferation on exogenous growth factors such as IL-2 and -15. Interestingly, CD8+CD45RA+CD27- cells parallel effector CTLs, as they abundantly express Fas-ligand mRNA, contain perforin and granzyme B, and have high cytolytic activity without in vitro prestimulation. Based on both phenotypic and functional properties, we conclude that memory- and effector-type T cells can be separated as distinct entities within the human CD8+ T cell subset.

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Intracellular measurement of cytokines in CD8+ T cells. Purified CD8+ cells were stimulated for 4 h (IFN-γ and IL-4) or 8 h (IL-2)  with PMA and ionomycin in the presence of the protein secretion inhibitor monensin. After surface staining with CD45 and CD27 mAbs, cells  were fixated and permeabilized and intracellular accumulated cytokines  were detected with specific mAbs. The histograms show the cytokine  staining. Dotted lines indicate the negative controls; the positive cell fraction is painted black; and the dot-plots show the distribution of positive  cells (black) among the total cell population (gray).
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Figure 3: Intracellular measurement of cytokines in CD8+ T cells. Purified CD8+ cells were stimulated for 4 h (IFN-γ and IL-4) or 8 h (IL-2) with PMA and ionomycin in the presence of the protein secretion inhibitor monensin. After surface staining with CD45 and CD27 mAbs, cells were fixated and permeabilized and intracellular accumulated cytokines were detected with specific mAbs. The histograms show the cytokine staining. Dotted lines indicate the negative controls; the positive cell fraction is painted black; and the dot-plots show the distribution of positive cells (black) among the total cell population (gray).

Mentions: In contrast to naive T cells, which primarily secrete IL-2, the ability to produce a variety of cytokines is a typical feature of primed T cells (35). Cytokine production capacity of CD8+ T cell subsets was measured after stimulation for 4 h with PMA and ionomycin at a single cell level (Fig. 3 and Table 2). CD8+CD45RA+CD27+ cells paralleled naive CD4+ cells in that IL-2 was the main cytokine produced by this subset. In agreement with what would be expected from primed cells, CD8+CD45RA− cells were able to secrete IL-2, IL-4, IFN-γ, and TNF-α. IL-4+ cells were exclusively found within this population with both CD27+ and CD27− cells contributing to the total IL-4 secretion. Also with respect to cytokine expression, the CD45RA+ CD27− subset showed characteristics of antigen-experienced cells, in that a high percentage of IFN-γ and TNF-α producers could be detected. Remarkably, in contrast to CD45RA− cells, neither IL-2 nor IL-4 production was found. Since IL-2 production in CD45RA+ cells has been described to reach its maximum later than in CD45RA− cells (36), we also analyzed 8-h stimulated cells. Indeed, the frequency of IL-2 producers increased in the CD45RA+ CD27+ subset. However, also at this time point, CD45RA+ CD27− cells did not produce any IL-2. From Fig. 3 it should be noted that, with respect to the cytokine secretion pattern, cells with a CD27dull expression behave as CD27− T cells in this assay. Furthermore, expression of surface markers on these cells was also comparable to CD27− cells (data not shown).


Phenotypic and functional separation of memory and effector human CD8+ T cells.

Hamann D, Baars PA, Rep MH, Hooibrink B, Kerkhof-Garde SR, Klein MR, van Lier RA - J. Exp. Med. (1997)

Intracellular measurement of cytokines in CD8+ T cells. Purified CD8+ cells were stimulated for 4 h (IFN-γ and IL-4) or 8 h (IL-2)  with PMA and ionomycin in the presence of the protein secretion inhibitor monensin. After surface staining with CD45 and CD27 mAbs, cells  were fixated and permeabilized and intracellular accumulated cytokines  were detected with specific mAbs. The histograms show the cytokine  staining. Dotted lines indicate the negative controls; the positive cell fraction is painted black; and the dot-plots show the distribution of positive  cells (black) among the total cell population (gray).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199103&req=5

Figure 3: Intracellular measurement of cytokines in CD8+ T cells. Purified CD8+ cells were stimulated for 4 h (IFN-γ and IL-4) or 8 h (IL-2) with PMA and ionomycin in the presence of the protein secretion inhibitor monensin. After surface staining with CD45 and CD27 mAbs, cells were fixated and permeabilized and intracellular accumulated cytokines were detected with specific mAbs. The histograms show the cytokine staining. Dotted lines indicate the negative controls; the positive cell fraction is painted black; and the dot-plots show the distribution of positive cells (black) among the total cell population (gray).
Mentions: In contrast to naive T cells, which primarily secrete IL-2, the ability to produce a variety of cytokines is a typical feature of primed T cells (35). Cytokine production capacity of CD8+ T cell subsets was measured after stimulation for 4 h with PMA and ionomycin at a single cell level (Fig. 3 and Table 2). CD8+CD45RA+CD27+ cells paralleled naive CD4+ cells in that IL-2 was the main cytokine produced by this subset. In agreement with what would be expected from primed cells, CD8+CD45RA− cells were able to secrete IL-2, IL-4, IFN-γ, and TNF-α. IL-4+ cells were exclusively found within this population with both CD27+ and CD27− cells contributing to the total IL-4 secretion. Also with respect to cytokine expression, the CD45RA+ CD27− subset showed characteristics of antigen-experienced cells, in that a high percentage of IFN-γ and TNF-α producers could be detected. Remarkably, in contrast to CD45RA− cells, neither IL-2 nor IL-4 production was found. Since IL-2 production in CD45RA+ cells has been described to reach its maximum later than in CD45RA− cells (36), we also analyzed 8-h stimulated cells. Indeed, the frequency of IL-2 producers increased in the CD45RA+ CD27+ subset. However, also at this time point, CD45RA+ CD27− cells did not produce any IL-2. From Fig. 3 it should be noted that, with respect to the cytokine secretion pattern, cells with a CD27dull expression behave as CD27− T cells in this assay. Furthermore, expression of surface markers on these cells was also comparable to CD27− cells (data not shown).

Bottom Line: These T cells do not secrete IL-2 or -4 but can produce IFN-gamma and TNF-alpha.Interestingly, CD8+CD45RA+CD27- cells parallel effector CTLs, as they abundantly express Fas-ligand mRNA, contain perforin and granzyme B, and have high cytolytic activity without in vitro prestimulation.Based on both phenotypic and functional properties, we conclude that memory- and effector-type T cells can be separated as distinct entities within the human CD8+ T cell subset.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Viro-Immunology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, University of Amsterdam.

ABSTRACT
Human CD8+ memory- and effector-type T cells are poorly defined. We show here that, next to a naive compartment, two discrete primed subpopulations can be found within the circulating human CD8+ T cell subset. First, CD45RA-CD45R0(+) cells are reminiscent of memory-type T cells in that they express elevated levels of CD95 (Fas) and the integrin family members CD11a, CD18, CD29, CD49d, and CD49e, compared to naive CD8+ T cells, and are able to secrete not only interleukin (IL) 2 but also interferon gamma, tumor necrosis factor alpha, and IL-4. This subset does not exert cytolytic activity without prior in vitro stimulation but does contain virus-specific cytotoxic T lymphocyte (CTL) precursors. A second primed population is characterized by CD45RA expression with concomitant absence of expression of the costimulatory molecules CD27 and CD28. The CD8+CD45RA+CD27- population contains T cells expressing high levels of CD11a, CD11b, CD18, and CD49d, whereas CD62L (L-selectin) is not expressed. These T cells do not secrete IL-2 or -4 but can produce IFN-gamma and TNF-alpha. In accordance with this finding, cells contained within this subpopulation depend for proliferation on exogenous growth factors such as IL-2 and -15. Interestingly, CD8+CD45RA+CD27- cells parallel effector CTLs, as they abundantly express Fas-ligand mRNA, contain perforin and granzyme B, and have high cytolytic activity without in vitro prestimulation. Based on both phenotypic and functional properties, we conclude that memory- and effector-type T cells can be separated as distinct entities within the human CD8+ T cell subset.

Show MeSH
Related in: MedlinePlus