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Antigen is required for the activation of effector activities, whereas interleukin 2 Is required for the maintenance of memory in ovalbumin-specific, CD8+ cytotoxic T lymphocytes.

Ke Y, Ma H, Kapp JA - J. Exp. Med. (1998)

Bottom Line: Thus, the effector functions of these CTLs were rapidly induced by T cell receptor (TCR) occupancy.In the absence of OVA, the precursor frequency was identical in spleens of normal and beta2-microglobulin knockout recipients, but significantly less in IL-2 knockout mice.The decline of memory in the absence of IL-2 supports data from other investigators, suggesting that cell cycling is important to the maintenance of CD8+ T cell memory.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Emory University School of Medicine, Atlanta, GA 30322, USA.

ABSTRACT
The mechanisms that maintain memory in T cells are not completely understood. We have investigated the role of antigen and interleukin (IL)-2 in the growth and maintenance of CD8+ T cells using a cytolytic T cell line specific for ovalbumin (OVA)257-264 presented by H-2Kb. This line does not secrete IL-4 or IL-2; hence, stimulation with the OVA-transfected EL4 line (E.G7-OVA) does not induce proliferation without addition of exogenous growth factors. Furthermore, this line can be maintained continuously by weekly addition of irradiated, splenic filler cells and IL-2, with or without E.G7-OVA. Although IL-2 induced proliferation of these cytotoxic T lymphocytes (CTLs), production of interferon gamma and tumor necrosis factor alpha required stimulation of the CTL with E. G7-OVA. The kinetics of lymphokine secretion after stimulation by E. G7-OVA were the same whether the CTL had been maintained with or without antigen (Ag). In addition, both CTL lines killed E.G7-OVA target cells within 4 h. Thus, the effector functions of these CTLs were rapidly induced by T cell receptor (TCR) occupancy. CTLs cultured with or without Ag also served as memory T cells when parked for 100 d in unirradiated, syngeneic recipients without OVA. In the absence of OVA, the precursor frequency was identical in spleens of normal and beta2-microglobulin knockout recipients, but significantly less in IL-2 knockout mice. The decline of memory in the absence of IL-2 supports data from other investigators, suggesting that cell cycling is important to the maintenance of CD8+ T cell memory. These data also suggest that stimulation of OVA-specific CTLs by lymphokines seems to be more important to maintaining memory than stimulation of TCRs by cross-reactive peptides complexed to class I molecules.

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Antigenic carryover  in OVA-CTLs. OVA-CTLs were  cultured in 24-well plates with  media, the indicated concentrations of irradiated E.G7-OVA, or  irradiated OVA-CTLs harvested  7 d after stimulation with irradiated E.G7-OVA. After overnight incubation, the supernatants were collected and analyzed  for IFN-γ production using an  ELISA with a recombinant IFN-γ  standard. The results are the  mean of triplicates ± SD from a  representative experiment repeated twice with similar results.
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Figure 7: Antigenic carryover in OVA-CTLs. OVA-CTLs were cultured in 24-well plates with media, the indicated concentrations of irradiated E.G7-OVA, or irradiated OVA-CTLs harvested 7 d after stimulation with irradiated E.G7-OVA. After overnight incubation, the supernatants were collected and analyzed for IFN-γ production using an ELISA with a recombinant IFN-γ standard. The results are the mean of triplicates ± SD from a representative experiment repeated twice with similar results.

Mentions: In contrast to naive animals, OVA-CTL lines can be stimulated to produce IFN-γ by irradiated E.G7-OVA, as illustrated in Fig. 1 B. This observation provides a sensitive method to gauge the amount of antigen remaining in the OVA-CTLs. 7 d after stimulation with E.G7-OVA, the OVA-CTLs were irradiated and added to fresh unirradiated OVA-CTLs. Activation was detected by secretion of IFN-γ. The results verify that OVA-CTLs do not produce IFN-γ without addition of exogenous antigen (Fig. 7), suggesting that little contaminating Ag remains in these cultures. No IFN-γ was produced by irradiated OVA-CTLs, nor did the irradiated OVA-CTLs stimulate IFN-γ production by unirradiated OVA-CTLs. Titration of irradiated E.G7-OVA established a standard curve for stimulation. From this experiment, we estimate that adoptive transfer of 5 × 106 OVA-CTLs would contain <5 × 103 cell equivalents of E.G7-OVA. In other words, <0.1% of the normal immunizing dose of E.G7-OVA might have been transferred as antigenic fragments to adoptive recipients. Hence, we conclude that the CTL activity recovered from B6 recipients of the OVA-CTLs is due to persistence of the CTLs as memory T cells rather than host CTLs primed by contaminating antigen.


Antigen is required for the activation of effector activities, whereas interleukin 2 Is required for the maintenance of memory in ovalbumin-specific, CD8+ cytotoxic T lymphocytes.

Ke Y, Ma H, Kapp JA - J. Exp. Med. (1998)

Antigenic carryover  in OVA-CTLs. OVA-CTLs were  cultured in 24-well plates with  media, the indicated concentrations of irradiated E.G7-OVA, or  irradiated OVA-CTLs harvested  7 d after stimulation with irradiated E.G7-OVA. After overnight incubation, the supernatants were collected and analyzed  for IFN-γ production using an  ELISA with a recombinant IFN-γ  standard. The results are the  mean of triplicates ± SD from a  representative experiment repeated twice with similar results.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199192&req=5

Figure 7: Antigenic carryover in OVA-CTLs. OVA-CTLs were cultured in 24-well plates with media, the indicated concentrations of irradiated E.G7-OVA, or irradiated OVA-CTLs harvested 7 d after stimulation with irradiated E.G7-OVA. After overnight incubation, the supernatants were collected and analyzed for IFN-γ production using an ELISA with a recombinant IFN-γ standard. The results are the mean of triplicates ± SD from a representative experiment repeated twice with similar results.
Mentions: In contrast to naive animals, OVA-CTL lines can be stimulated to produce IFN-γ by irradiated E.G7-OVA, as illustrated in Fig. 1 B. This observation provides a sensitive method to gauge the amount of antigen remaining in the OVA-CTLs. 7 d after stimulation with E.G7-OVA, the OVA-CTLs were irradiated and added to fresh unirradiated OVA-CTLs. Activation was detected by secretion of IFN-γ. The results verify that OVA-CTLs do not produce IFN-γ without addition of exogenous antigen (Fig. 7), suggesting that little contaminating Ag remains in these cultures. No IFN-γ was produced by irradiated OVA-CTLs, nor did the irradiated OVA-CTLs stimulate IFN-γ production by unirradiated OVA-CTLs. Titration of irradiated E.G7-OVA established a standard curve for stimulation. From this experiment, we estimate that adoptive transfer of 5 × 106 OVA-CTLs would contain <5 × 103 cell equivalents of E.G7-OVA. In other words, <0.1% of the normal immunizing dose of E.G7-OVA might have been transferred as antigenic fragments to adoptive recipients. Hence, we conclude that the CTL activity recovered from B6 recipients of the OVA-CTLs is due to persistence of the CTLs as memory T cells rather than host CTLs primed by contaminating antigen.

Bottom Line: Thus, the effector functions of these CTLs were rapidly induced by T cell receptor (TCR) occupancy.In the absence of OVA, the precursor frequency was identical in spleens of normal and beta2-microglobulin knockout recipients, but significantly less in IL-2 knockout mice.The decline of memory in the absence of IL-2 supports data from other investigators, suggesting that cell cycling is important to the maintenance of CD8+ T cell memory.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Emory University School of Medicine, Atlanta, GA 30322, USA.

ABSTRACT
The mechanisms that maintain memory in T cells are not completely understood. We have investigated the role of antigen and interleukin (IL)-2 in the growth and maintenance of CD8+ T cells using a cytolytic T cell line specific for ovalbumin (OVA)257-264 presented by H-2Kb. This line does not secrete IL-4 or IL-2; hence, stimulation with the OVA-transfected EL4 line (E.G7-OVA) does not induce proliferation without addition of exogenous growth factors. Furthermore, this line can be maintained continuously by weekly addition of irradiated, splenic filler cells and IL-2, with or without E.G7-OVA. Although IL-2 induced proliferation of these cytotoxic T lymphocytes (CTLs), production of interferon gamma and tumor necrosis factor alpha required stimulation of the CTL with E. G7-OVA. The kinetics of lymphokine secretion after stimulation by E. G7-OVA were the same whether the CTL had been maintained with or without antigen (Ag). In addition, both CTL lines killed E.G7-OVA target cells within 4 h. Thus, the effector functions of these CTLs were rapidly induced by T cell receptor (TCR) occupancy. CTLs cultured with or without Ag also served as memory T cells when parked for 100 d in unirradiated, syngeneic recipients without OVA. In the absence of OVA, the precursor frequency was identical in spleens of normal and beta2-microglobulin knockout recipients, but significantly less in IL-2 knockout mice. The decline of memory in the absence of IL-2 supports data from other investigators, suggesting that cell cycling is important to the maintenance of CD8+ T cell memory. These data also suggest that stimulation of OVA-specific CTLs by lymphokines seems to be more important to maintaining memory than stimulation of TCRs by cross-reactive peptides complexed to class I molecules.

Show MeSH
Related in: MedlinePlus