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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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Metabolic inhibitors  prevent cytokine production,  but not cytolytic activity of  CTLs. OVA-CTLs (106/ml)  were pretreated with 5 μg/ml  actinomycin D, 15 μg/ml cycloheximide, or medium at 37°C  for 1 h. The cells were then  washed and incubated with 105  of E.G7-OVA. After incubation  at 37°C for various periods of  time, supernatants were tested  for production of (A) IFN-γ and  (B) TNF-α by ELISA. In C,  pretreated OVA-CTLs were incubated with 51Cr-labeled targets. After 4 h, supernatant fluids  were collected and counted. The  results of one of two similar experiments are shown as the mean  of triplicates ± SD.
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Figure 5: Metabolic inhibitors prevent cytokine production, but not cytolytic activity of CTLs. OVA-CTLs (106/ml) were pretreated with 5 μg/ml actinomycin D, 15 μg/ml cycloheximide, or medium at 37°C for 1 h. The cells were then washed and incubated with 105 of E.G7-OVA. After incubation at 37°C for various periods of time, supernatants were tested for production of (A) IFN-γ and (B) TNF-α by ELISA. In C, pretreated OVA-CTLs were incubated with 51Cr-labeled targets. After 4 h, supernatant fluids were collected and counted. The results of one of two similar experiments are shown as the mean of triplicates ± SD.

Mentions: The rapidity with which TNF-α and IFN-γ were secreted into the supernates of the CTLs after stimulation with Ags (Fig. 4) raised a question about whether the secreted lymphokines were newly synthesized or released from preexisting stores. However, cycloheximide inhibited the secretion of IFN-γ (Fig. 5 A) and TNF-α (Fig. 5 B) stimulated by E.G7-OVA, suggesting that these proteins are synthesized in response to antigenic stimulation. The finding that actinomycin D also inhibited secretion of the lymphokines (Fig. 5) suggests that new messenger RNA synthesis was also required for lymphokine secretion. Thus, cultured OVA-specific CTLs upregulate lymphokine gene expression within minutes after stimulation by antigen. By contrast, lysis of targets by OVA-specific CTLs was relatively insensitive to inhibitors of protein and RNA synthesis (Fig. 5 C) as would be expected because the lytic proteins, perforin and granzymes, are stored in granules for release upon target cell recognition.


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)

Metabolic inhibitors  prevent cytokine production,  but not cytolytic activity of  CTLs. OVA-CTLs (106/ml)  were pretreated with 5 μg/ml  actinomycin D, 15 μg/ml cycloheximide, or medium at 37°C  for 1 h. The cells were then  washed and incubated with 105  of E.G7-OVA. After incubation  at 37°C for various periods of  time, supernatants were tested  for production of (A) IFN-γ and  (B) TNF-α by ELISA. In C,  pretreated OVA-CTLs were incubated with 51Cr-labeled targets. After 4 h, supernatant fluids  were collected and counted. The  results of one of two similar experiments are shown as the mean  of triplicates ± SD.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199192&req=5

Figure 5: Metabolic inhibitors prevent cytokine production, but not cytolytic activity of CTLs. OVA-CTLs (106/ml) were pretreated with 5 μg/ml actinomycin D, 15 μg/ml cycloheximide, or medium at 37°C for 1 h. The cells were then washed and incubated with 105 of E.G7-OVA. After incubation at 37°C for various periods of time, supernatants were tested for production of (A) IFN-γ and (B) TNF-α by ELISA. In C, pretreated OVA-CTLs were incubated with 51Cr-labeled targets. After 4 h, supernatant fluids were collected and counted. The results of one of two similar experiments are shown as the mean of triplicates ± SD.
Mentions: The rapidity with which TNF-α and IFN-γ were secreted into the supernates of the CTLs after stimulation with Ags (Fig. 4) raised a question about whether the secreted lymphokines were newly synthesized or released from preexisting stores. However, cycloheximide inhibited the secretion of IFN-γ (Fig. 5 A) and TNF-α (Fig. 5 B) stimulated by E.G7-OVA, suggesting that these proteins are synthesized in response to antigenic stimulation. The finding that actinomycin D also inhibited secretion of the lymphokines (Fig. 5) suggests that new messenger RNA synthesis was also required for lymphokine secretion. Thus, cultured OVA-specific CTLs upregulate lymphokine gene expression within minutes after stimulation by antigen. By contrast, lysis of targets by OVA-specific CTLs was relatively insensitive to inhibitors of protein and RNA synthesis (Fig. 5 C) as would be expected because the lytic proteins, perforin and granzymes, are stored in granules for release upon target cell recognition.

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