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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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Comparison of cytokine production between stimulated and  unstimulated CTLs. Stimulated OVA-CTLs were stimulated weekly with  irradiated E.G7-OVA, syngeneic filler cells, and Con A supernatant. Unstimulated OVA-CTLs were maintained by weekly replenishment with  irradiated filler cells and Con A supernatant, but without E.G7-OVA. After 100 d, 106 of stimulated or unstimulated OVA-CTLs were incubated  with 105 of EL4 or E.G7-OVA, or without targets. After incubation at  37°C, supernatants were tested for production of (A) IFN-γ and (B)  TNF-α by ELISA. The results shown are the mean of triplicates ± SD of  a representative experiment that has been repeated twice.
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Figure 4: Comparison of cytokine production between stimulated and unstimulated CTLs. Stimulated OVA-CTLs were stimulated weekly with irradiated E.G7-OVA, syngeneic filler cells, and Con A supernatant. Unstimulated OVA-CTLs were maintained by weekly replenishment with irradiated filler cells and Con A supernatant, but without E.G7-OVA. After 100 d, 106 of stimulated or unstimulated OVA-CTLs were incubated with 105 of EL4 or E.G7-OVA, or without targets. After incubation at 37°C, supernatants were tested for production of (A) IFN-γ and (B) TNF-α by ELISA. The results shown are the mean of triplicates ± SD of a representative experiment that has been repeated twice.

Mentions: We also investigated whether there were any differences in cytokine production between these OVA-CTL lines maintained with or without Ags. OVA-CTLs were incubated with or without stimulator cells. Supernatants were collected at various times and tested for IFN-γ or TNF-α production by ELISA. A latent phase of 1–2 h was observed before IFN-γ (Fig. 4 A) or TNF-α (Fig. 4 B) were detected in the supernates of the OVA-CTLs that had been stimulated weekly with E.G7-OVA. Activation of the OVA-CTLs that had been maintained with filler cells and IL-2 occurred a bit later and with slightly slower kinetics than seen with OVA-CTLs maintained with Ag. However, comparable levels of IFN-γ or TNF-α were produced by both cultures of OVA-CTLs after 4 h of incubation with E.G7-OVA. Neither of the cultures produced lymphokines after stimulation by EL4. These results confirm that OVA-CTLs, cultured with filler cells and IL-2 for long periods of time, retained their biological functions as well as their antigenic specificity.


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)

Comparison of cytokine production between stimulated and  unstimulated CTLs. Stimulated OVA-CTLs were stimulated weekly with  irradiated E.G7-OVA, syngeneic filler cells, and Con A supernatant. Unstimulated OVA-CTLs were maintained by weekly replenishment with  irradiated filler cells and Con A supernatant, but without E.G7-OVA. After 100 d, 106 of stimulated or unstimulated OVA-CTLs were incubated  with 105 of EL4 or E.G7-OVA, or without targets. After incubation at  37°C, supernatants were tested for production of (A) IFN-γ and (B)  TNF-α by ELISA. The results shown are the mean of triplicates ± SD of  a representative experiment that has been repeated twice.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Comparison of cytokine production between stimulated and unstimulated CTLs. Stimulated OVA-CTLs were stimulated weekly with irradiated E.G7-OVA, syngeneic filler cells, and Con A supernatant. Unstimulated OVA-CTLs were maintained by weekly replenishment with irradiated filler cells and Con A supernatant, but without E.G7-OVA. After 100 d, 106 of stimulated or unstimulated OVA-CTLs were incubated with 105 of EL4 or E.G7-OVA, or without targets. After incubation at 37°C, supernatants were tested for production of (A) IFN-γ and (B) TNF-α by ELISA. The results shown are the mean of triplicates ± SD of a representative experiment that has been repeated twice.
Mentions: We also investigated whether there were any differences in cytokine production between these OVA-CTL lines maintained with or without Ags. OVA-CTLs were incubated with or without stimulator cells. Supernatants were collected at various times and tested for IFN-γ or TNF-α production by ELISA. A latent phase of 1–2 h was observed before IFN-γ (Fig. 4 A) or TNF-α (Fig. 4 B) were detected in the supernates of the OVA-CTLs that had been stimulated weekly with E.G7-OVA. Activation of the OVA-CTLs that had been maintained with filler cells and IL-2 occurred a bit later and with slightly slower kinetics than seen with OVA-CTLs maintained with Ag. However, comparable levels of IFN-γ or TNF-α were produced by both cultures of OVA-CTLs after 4 h of incubation with E.G7-OVA. Neither of the cultures produced lymphokines after stimulation by EL4. These results confirm that OVA-CTLs, cultured with filler cells and IL-2 for long periods of time, retained their biological functions as well as their antigenic specificity.

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