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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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Persistence of donor CTLs in recipients. Unirradiated, syngeneic mice were injected intravenously with 107 (A) OVA-CTLs or (B)  allogeneic B10 anti-BALB/c CTLs. After 100 d, recipients were killed  and spleen cells were titrated in 96-well plates. Irradiated E.G7-OVA,  syngeneic filler cells, and Con A supernatant were added to the cultures.  After incubation at 37°C for 1 wk, the cytolytic activity of half of the  wells was tested using 51Cr-labeled E.G7-OVA targets. The other half of  the cultures were restimulated as described above and tested for cytolytic  activity after an additional week in culture. Results shown are the averages of 12 wells ± SD.
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Figure 6: Persistence of donor CTLs in recipients. Unirradiated, syngeneic mice were injected intravenously with 107 (A) OVA-CTLs or (B) allogeneic B10 anti-BALB/c CTLs. After 100 d, recipients were killed and spleen cells were titrated in 96-well plates. Irradiated E.G7-OVA, syngeneic filler cells, and Con A supernatant were added to the cultures. After incubation at 37°C for 1 wk, the cytolytic activity of half of the wells was tested using 51Cr-labeled E.G7-OVA targets. The other half of the cultures were restimulated as described above and tested for cytolytic activity after an additional week in culture. Results shown are the averages of 12 wells ± SD.

Mentions: CTL lines that are stimulated weekly with antigen and/or IL-2 do not revert to a resting state. Such cell lines should be considered to be effector cells rather than memory T cells. To test whether such CTL lines could persist in vivo in the absence of an antigenic stimulus, OVA-CTLs were harvested 7 d after stimulation with irradiated E.G7-OVA. No viable E.G7-OVA remain in these cultures, as shown by the failure of these cultures to stimulate fresh CTLs (not shown). Dead cells and debris were removed from the CTLs by Ficoll-Hypaque centrifugation. Viable OVA-CTLs and B10 α BALB/c CTLs were adoptively transferred to normal, syngeneic mice without secondary challenge. To provide a physiological environment, the recipient animals were not irradiated. Spleen cells from these recipients were harvested 100 d after transfer and stimulated in vitro with E.G7-OVA. After stimulation for 1 and 2 wk, OVA-specific cytolytic activities were detected in spleen cells from mice receiving OVA-specific CTLs (Fig. 6 A). Spleen cells from mice receiving control B10 α BALB/c CTLs did not develop OVA-specific responses (Fig. 6 B), which is consistent with the previous reports that OVA-specific CTLs cannot be induced by stimulation with E.G7-OVA in vitro without previous priming (9). These results suggest that CTLs persisted in vivo in the absence of viable antigen-bearing tumor cells.


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)

Persistence of donor CTLs in recipients. Unirradiated, syngeneic mice were injected intravenously with 107 (A) OVA-CTLs or (B)  allogeneic B10 anti-BALB/c CTLs. After 100 d, recipients were killed  and spleen cells were titrated in 96-well plates. Irradiated E.G7-OVA,  syngeneic filler cells, and Con A supernatant were added to the cultures.  After incubation at 37°C for 1 wk, the cytolytic activity of half of the  wells was tested using 51Cr-labeled E.G7-OVA targets. The other half of  the cultures were restimulated as described above and tested for cytolytic  activity after an additional week in culture. Results shown are the averages of 12 wells ± SD.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199192&req=5

Figure 6: Persistence of donor CTLs in recipients. Unirradiated, syngeneic mice were injected intravenously with 107 (A) OVA-CTLs or (B) allogeneic B10 anti-BALB/c CTLs. After 100 d, recipients were killed and spleen cells were titrated in 96-well plates. Irradiated E.G7-OVA, syngeneic filler cells, and Con A supernatant were added to the cultures. After incubation at 37°C for 1 wk, the cytolytic activity of half of the wells was tested using 51Cr-labeled E.G7-OVA targets. The other half of the cultures were restimulated as described above and tested for cytolytic activity after an additional week in culture. Results shown are the averages of 12 wells ± SD.
Mentions: CTL lines that are stimulated weekly with antigen and/or IL-2 do not revert to a resting state. Such cell lines should be considered to be effector cells rather than memory T cells. To test whether such CTL lines could persist in vivo in the absence of an antigenic stimulus, OVA-CTLs were harvested 7 d after stimulation with irradiated E.G7-OVA. No viable E.G7-OVA remain in these cultures, as shown by the failure of these cultures to stimulate fresh CTLs (not shown). Dead cells and debris were removed from the CTLs by Ficoll-Hypaque centrifugation. Viable OVA-CTLs and B10 α BALB/c CTLs were adoptively transferred to normal, syngeneic mice without secondary challenge. To provide a physiological environment, the recipient animals were not irradiated. Spleen cells from these recipients were harvested 100 d after transfer and stimulated in vitro with E.G7-OVA. After stimulation for 1 and 2 wk, OVA-specific cytolytic activities were detected in spleen cells from mice receiving OVA-specific CTLs (Fig. 6 A). Spleen cells from mice receiving control B10 α BALB/c CTLs did not develop OVA-specific responses (Fig. 6 B), which is consistent with the previous reports that OVA-specific CTLs cannot be induced by stimulation with E.G7-OVA in vitro without previous priming (9). These results suggest that CTLs persisted in vivo in the absence of viable antigen-bearing tumor cells.

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