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Phenotypic and functional analysis of CD8(+) T cells undergoing peripheral deletion in response to cross-presentation of self-antigen.

Hernandez J, Aung S, Redmond WL, Sherman LA - J. Exp. Med. (2001)

Bottom Line: Not all T cells specific for autoantigens are eliminated in the thymus, and therefore alternate mechanisms are required to prevent potentially autoreactive T cells from developing into effectors.The cells undergoing tolerance upregulate both CD69 and CD44, yet only partially downregulate CD62L, and do not express CD49d or CD25.These data demonstrate that the recognition by naive CD8(+) T cells of cross-presented self-antigen results in localized proliferation and deletion, without the production of effector cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
Not all T cells specific for autoantigens are eliminated in the thymus, and therefore alternate mechanisms are required to prevent potentially autoreactive T cells from developing into effectors. Adoptive transfer of CD8(+) T cells from influenza hemagglutinin-specific Clone 4 TCR transgenic mice into mice that express hemagluttinin in the pancreatic islets results in tolerance. This is preceded by activation of Clone 4 T cells that encounter antigen cross-presented in the draining lymph nodes of the pancreas. In this report we compare the phenotype, function, and costimulatory requirements of Clone 4 T cells activated by endogenous self-antigen, with Clone 4 T cells stimulated by influenza virus. The cells undergoing tolerance upregulate both CD69 and CD44, yet only partially downregulate CD62L, and do not express CD49d or CD25. Most importantly, they lack the ability to produce interferon-gamma in response to antigen and show no cytolytic activity. Clone 4 T cells disappear after several cycles of division, apparently without leaving the site of initial activation. Surprisingly, despite the fact that such stimulation occurs through recognition of antigen that is cross-presented by a professional antigen-presenting cell, we find this activation is not dependent on costimulation through CD28. These data demonstrate that the recognition by naive CD8(+) T cells of cross-presented self-antigen results in localized proliferation and deletion, without the production of effector cells.

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IFN-γ production by proliferating Clone 4 CD8+ T cells. 3 × 106 CFSE-labeled, purified, Thy1.1+ Clone 4 CD8+ T cells were injected into InsHA recipients that were either immunized with influenza virus or left untreated. On day 4 after transfer cells from pooled pancreatic LNs were incubated with Kd HA peptide (stimulated) or an irrelevant peptide (nonstimulated) for 6 h at 37°C. Then, cells were analyzed by FACS® to detect accumulation of intracellular IFN-γ. (A) Plots represent the amount of CFSE label versus the intensity of IFN-γ produced gating on lymphocytes CD8+ Thy1.1+. Data correspond to one out of two independent experiments with similar results. (B) Representation of the percentage of CD8+ Thy1.1+ T cells that are IFN-γ+ from A as a function of the division cycle.
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Figure 4: IFN-γ production by proliferating Clone 4 CD8+ T cells. 3 × 106 CFSE-labeled, purified, Thy1.1+ Clone 4 CD8+ T cells were injected into InsHA recipients that were either immunized with influenza virus or left untreated. On day 4 after transfer cells from pooled pancreatic LNs were incubated with Kd HA peptide (stimulated) or an irrelevant peptide (nonstimulated) for 6 h at 37°C. Then, cells were analyzed by FACS® to detect accumulation of intracellular IFN-γ. (A) Plots represent the amount of CFSE label versus the intensity of IFN-γ produced gating on lymphocytes CD8+ Thy1.1+. Data correspond to one out of two independent experiments with similar results. (B) Representation of the percentage of CD8+ Thy1.1+ T cells that are IFN-γ+ from A as a function of the division cycle.

Mentions: The observation that Clone 4 CD8+ T cells stimulated by endogenous self-antigen did not express a “classical” effector CTL phenotype led us to question whether the activated cells demonstrated effector function. Production of IFN-γ in response to antigen represents an early effector function. Indeed, a total of >60% of the Clone 4 cells obtained from influenza-infected mice exhibited the ability to produce IFN-γ at day 4 after transfer (Fig. 4). This cytokine was clearly detected even in cells that had divided only once or twice (Fig. 4). In contrast, the Clone 4 T cells taken from the pancreatic LNs of InsHA mice did not produce IFN-γ at day 4 after transfer (Fig. 4). Due to the limiting amount of cross-presented antigen few of the Clone 4 T cells transferred into InsHA are activated and proliferate at any given time. This is an ongoing process that continues until all naive Clone 4 cells are tolerized 13. Therefore, the cells that have experienced fewer rounds of division are likely to be derived from naive cells that were more recently activated. Accordingly, these data argues against the possibility that cells undergoing tolerance may produce IFN-γ at an early stage in division. In addition, examination of the Clone 4 cells taken from InsHA mice at day 2 after transfer, a time point when they have only divided one or two times, also showed no production of IFN-γ (data not shown).


Phenotypic and functional analysis of CD8(+) T cells undergoing peripheral deletion in response to cross-presentation of self-antigen.

Hernandez J, Aung S, Redmond WL, Sherman LA - J. Exp. Med. (2001)

IFN-γ production by proliferating Clone 4 CD8+ T cells. 3 × 106 CFSE-labeled, purified, Thy1.1+ Clone 4 CD8+ T cells were injected into InsHA recipients that were either immunized with influenza virus or left untreated. On day 4 after transfer cells from pooled pancreatic LNs were incubated with Kd HA peptide (stimulated) or an irrelevant peptide (nonstimulated) for 6 h at 37°C. Then, cells were analyzed by FACS® to detect accumulation of intracellular IFN-γ. (A) Plots represent the amount of CFSE label versus the intensity of IFN-γ produced gating on lymphocytes CD8+ Thy1.1+. Data correspond to one out of two independent experiments with similar results. (B) Representation of the percentage of CD8+ Thy1.1+ T cells that are IFN-γ+ from A as a function of the division cycle.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195957&req=5

Figure 4: IFN-γ production by proliferating Clone 4 CD8+ T cells. 3 × 106 CFSE-labeled, purified, Thy1.1+ Clone 4 CD8+ T cells were injected into InsHA recipients that were either immunized with influenza virus or left untreated. On day 4 after transfer cells from pooled pancreatic LNs were incubated with Kd HA peptide (stimulated) or an irrelevant peptide (nonstimulated) for 6 h at 37°C. Then, cells were analyzed by FACS® to detect accumulation of intracellular IFN-γ. (A) Plots represent the amount of CFSE label versus the intensity of IFN-γ produced gating on lymphocytes CD8+ Thy1.1+. Data correspond to one out of two independent experiments with similar results. (B) Representation of the percentage of CD8+ Thy1.1+ T cells that are IFN-γ+ from A as a function of the division cycle.
Mentions: The observation that Clone 4 CD8+ T cells stimulated by endogenous self-antigen did not express a “classical” effector CTL phenotype led us to question whether the activated cells demonstrated effector function. Production of IFN-γ in response to antigen represents an early effector function. Indeed, a total of >60% of the Clone 4 cells obtained from influenza-infected mice exhibited the ability to produce IFN-γ at day 4 after transfer (Fig. 4). This cytokine was clearly detected even in cells that had divided only once or twice (Fig. 4). In contrast, the Clone 4 T cells taken from the pancreatic LNs of InsHA mice did not produce IFN-γ at day 4 after transfer (Fig. 4). Due to the limiting amount of cross-presented antigen few of the Clone 4 T cells transferred into InsHA are activated and proliferate at any given time. This is an ongoing process that continues until all naive Clone 4 cells are tolerized 13. Therefore, the cells that have experienced fewer rounds of division are likely to be derived from naive cells that were more recently activated. Accordingly, these data argues against the possibility that cells undergoing tolerance may produce IFN-γ at an early stage in division. In addition, examination of the Clone 4 cells taken from InsHA mice at day 2 after transfer, a time point when they have only divided one or two times, also showed no production of IFN-γ (data not shown).

Bottom Line: Not all T cells specific for autoantigens are eliminated in the thymus, and therefore alternate mechanisms are required to prevent potentially autoreactive T cells from developing into effectors.The cells undergoing tolerance upregulate both CD69 and CD44, yet only partially downregulate CD62L, and do not express CD49d or CD25.These data demonstrate that the recognition by naive CD8(+) T cells of cross-presented self-antigen results in localized proliferation and deletion, without the production of effector cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
Not all T cells specific for autoantigens are eliminated in the thymus, and therefore alternate mechanisms are required to prevent potentially autoreactive T cells from developing into effectors. Adoptive transfer of CD8(+) T cells from influenza hemagglutinin-specific Clone 4 TCR transgenic mice into mice that express hemagluttinin in the pancreatic islets results in tolerance. This is preceded by activation of Clone 4 T cells that encounter antigen cross-presented in the draining lymph nodes of the pancreas. In this report we compare the phenotype, function, and costimulatory requirements of Clone 4 T cells activated by endogenous self-antigen, with Clone 4 T cells stimulated by influenza virus. The cells undergoing tolerance upregulate both CD69 and CD44, yet only partially downregulate CD62L, and do not express CD49d or CD25. Most importantly, they lack the ability to produce interferon-gamma in response to antigen and show no cytolytic activity. Clone 4 T cells disappear after several cycles of division, apparently without leaving the site of initial activation. Surprisingly, despite the fact that such stimulation occurs through recognition of antigen that is cross-presented by a professional antigen-presenting cell, we find this activation is not dependent on costimulation through CD28. These data demonstrate that the recognition by naive CD8(+) T cells of cross-presented self-antigen results in localized proliferation and deletion, without the production of effector cells.

Show MeSH
Related in: MedlinePlus