Limits...
A sufficient role of MHC class I molecules on hepatocytes in anti-plasmodial activity of CD8 (+) T cells in vivo.

Huang J, Tsao T, Zhang M, Rai U, Tsuji M, Li X - Front Microbiol (2015)

Bottom Line: Presently, there is only one CD8(+) epitope, SYVPSAEQI, derived from the circumsporozoite protein of Plasmodium yoelii (PyCS), that mediates anti-malarial protection and is presented in the context of a K(d) molecule.We found that the adoptive transfer of a PyCS-specific CD8(+) T-cell line resulted in a significant inhibition of the parasite burden in the liver of Alb-K(d) Tg, as well as MHC-I-K(d) Tg mice, but not of C57BL/6 mice.These results indicate that the K(d) molecule expressed by hepatocytes is sufficient in mediating the anti-plasmodial activity of PyCS-specific CD8(+) T cells in vivo.

View Article: PubMed Central - PubMed

Affiliation: HIV and Malaria Vaccine Program, Aaron Diamond AIDS Research Center - The Rockefeller University New York, NY, USA.

ABSTRACT
Although CD8(+) T cells are shown to mediate the protective immunity against the liver stages of malaria parasites in mice, whether the direct presentation of malaria antigen by major histocompatibility complex (MHC) class I molecules expressed on the liver of infected host is required for anti-plasmodial activity of CD8(+) T cells is still unknown. Presently, there is only one CD8(+) epitope, SYVPSAEQI, derived from the circumsporozoite protein of Plasmodium yoelii (PyCS), that mediates anti-malarial protection and is presented in the context of a K(d) molecule. Therefore, to investigate the mode of anti-plasmodial activity of CD8+ T cells, we have previously generated C57BL/6 transgenic (Tg) mice, in which a K(d) molecule is expressed only on hepatocyte (Alb-K(d)) or dendritic cell (DC; CD11c-K(d)), by using albumin promoter or CD11c promoter, respectively. We have also generated MHC-I-K(d) Tg mice, which express the K(d) molecule under the MHC class I (MHC-I) promoter, as a positive control. From splenocytes collected from CD11c-K(d) Tg mice immunized with a synthetic peptide, SYVPSAEQI, which corresponds to the CD8(+) T-cell epitope of PyCS, emulsified in incomplete Freund's adjuvant , a PyCS-specific CD8(+) T-cell line was generated. This PyCS-specific CD8(+)T-cell line was then adoptively transferred into a cohort of either MHC-K(d) Tg or Alb-K(d) Tg mice listed above, as well as wild-type C57BL/6 mice. Then both transferred and non-transferred mice were challenged with live malaria parasites. We found that the adoptive transfer of a PyCS-specific CD8(+) T-cell line resulted in a significant inhibition of the parasite burden in the liver of Alb-K(d) Tg, as well as MHC-I-K(d) Tg mice, but not of C57BL/6 mice. These results indicate that the K(d) molecule expressed by hepatocytes is sufficient in mediating the anti-plasmodial activity of PyCS-specific CD8(+) T cells in vivo.

No MeSH data available.


Related in: MedlinePlus

Frequency of a PyCS-specific CD8+ T-cell line, as determined by either a tetramer staining or an ELISpot assay. Frequency of PyCS-specific CD8+ T cells among the T-cell line was determined by either a tetramer staining (A) or an IFN-γELISpot assay (B). In (A), CD3+ T cells gated with anti-CD8 antibody were stained with APC-labeled H-2Kd/SYVPSAEQI-tetramer. The number shows the percentage of CD8+ T cells that are positive with H-2Kd/SYVPSAEQI-tetramer. In (B), 1000 cells of the T-cell line were co-cultured with 5 × 105 EL-4-Kd cells loaded with SYVPSAEQI in a well of an ELISpot plate, and 24 h later, the relative number of IFN-γ-secreting cells was determined by an ELISpot assay. The number of IFN-γ-secreting cells after co-culturing with EL-4-Kd cells without SYVPSAEQI peptide loading was used as a negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4325910&req=5

Figure 1: Frequency of a PyCS-specific CD8+ T-cell line, as determined by either a tetramer staining or an ELISpot assay. Frequency of PyCS-specific CD8+ T cells among the T-cell line was determined by either a tetramer staining (A) or an IFN-γELISpot assay (B). In (A), CD3+ T cells gated with anti-CD8 antibody were stained with APC-labeled H-2Kd/SYVPSAEQI-tetramer. The number shows the percentage of CD8+ T cells that are positive with H-2Kd/SYVPSAEQI-tetramer. In (B), 1000 cells of the T-cell line were co-cultured with 5 × 105 EL-4-Kd cells loaded with SYVPSAEQI in a well of an ELISpot plate, and 24 h later, the relative number of IFN-γ-secreting cells was determined by an ELISpot assay. The number of IFN-γ-secreting cells after co-culturing with EL-4-Kd cells without SYVPSAEQI peptide loading was used as a negative control.

Mentions: CD11c-Kd Tg mice were immunized twice with SYVPSAEQI peptide emulsified in IFA with a 2-week interval. Then splenocytes from peptide-immunized CD11c-Kd Tg mice were collected for the expansion of a PyCS-specific CD8+ T-cell line in vitro, which was achieved by stimulating the cells for a few times with irradiated MHC-I-Kd Tg mouse-derived splenocytes pulsed with the SYVPSAEQI peptide in a 10–14-day interval, as similarly performed previously (Rodrigues et al., 1991). After successful expansion of a PyCS-specific CD8+ T-cell line, the specificity and frequency of the PyCS-specific CD8+ T-cell line were determined by either APC-labeled H-2Kd/SYVPSAEQI-tetramer staining or IFN-γ ELISpot assay, as shown in Figure 1. The tetramer staining results depicted that more than two third of the cells of a PyCS-specific CD8+ T-cell line could be identified as H-2Kd/SYVPSAEQI tetramer CD8+ T cells (Figure 1A). By assessing the ELISpot assay, more than 700 cells out of 1,000 cells of the T cell line were found to get activated by the SYVPSAEQI peptide and secrete IFN-γ, whereas in the absence of the SYVPSAEQI peptide, the cells did not display any responses, indicating that there was no non-specific or auto-reactive T cells present in the T cell line (Figure 1B). This indicates that a majority (>65–70%) of the population in the T cell line are specific to the SYVPSAEQI epitope.


A sufficient role of MHC class I molecules on hepatocytes in anti-plasmodial activity of CD8 (+) T cells in vivo.

Huang J, Tsao T, Zhang M, Rai U, Tsuji M, Li X - Front Microbiol (2015)

Frequency of a PyCS-specific CD8+ T-cell line, as determined by either a tetramer staining or an ELISpot assay. Frequency of PyCS-specific CD8+ T cells among the T-cell line was determined by either a tetramer staining (A) or an IFN-γELISpot assay (B). In (A), CD3+ T cells gated with anti-CD8 antibody were stained with APC-labeled H-2Kd/SYVPSAEQI-tetramer. The number shows the percentage of CD8+ T cells that are positive with H-2Kd/SYVPSAEQI-tetramer. In (B), 1000 cells of the T-cell line were co-cultured with 5 × 105 EL-4-Kd cells loaded with SYVPSAEQI in a well of an ELISpot plate, and 24 h later, the relative number of IFN-γ-secreting cells was determined by an ELISpot assay. The number of IFN-γ-secreting cells after co-culturing with EL-4-Kd cells without SYVPSAEQI peptide loading was used as a negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4325910&req=5

Figure 1: Frequency of a PyCS-specific CD8+ T-cell line, as determined by either a tetramer staining or an ELISpot assay. Frequency of PyCS-specific CD8+ T cells among the T-cell line was determined by either a tetramer staining (A) or an IFN-γELISpot assay (B). In (A), CD3+ T cells gated with anti-CD8 antibody were stained with APC-labeled H-2Kd/SYVPSAEQI-tetramer. The number shows the percentage of CD8+ T cells that are positive with H-2Kd/SYVPSAEQI-tetramer. In (B), 1000 cells of the T-cell line were co-cultured with 5 × 105 EL-4-Kd cells loaded with SYVPSAEQI in a well of an ELISpot plate, and 24 h later, the relative number of IFN-γ-secreting cells was determined by an ELISpot assay. The number of IFN-γ-secreting cells after co-culturing with EL-4-Kd cells without SYVPSAEQI peptide loading was used as a negative control.
Mentions: CD11c-Kd Tg mice were immunized twice with SYVPSAEQI peptide emulsified in IFA with a 2-week interval. Then splenocytes from peptide-immunized CD11c-Kd Tg mice were collected for the expansion of a PyCS-specific CD8+ T-cell line in vitro, which was achieved by stimulating the cells for a few times with irradiated MHC-I-Kd Tg mouse-derived splenocytes pulsed with the SYVPSAEQI peptide in a 10–14-day interval, as similarly performed previously (Rodrigues et al., 1991). After successful expansion of a PyCS-specific CD8+ T-cell line, the specificity and frequency of the PyCS-specific CD8+ T-cell line were determined by either APC-labeled H-2Kd/SYVPSAEQI-tetramer staining or IFN-γ ELISpot assay, as shown in Figure 1. The tetramer staining results depicted that more than two third of the cells of a PyCS-specific CD8+ T-cell line could be identified as H-2Kd/SYVPSAEQI tetramer CD8+ T cells (Figure 1A). By assessing the ELISpot assay, more than 700 cells out of 1,000 cells of the T cell line were found to get activated by the SYVPSAEQI peptide and secrete IFN-γ, whereas in the absence of the SYVPSAEQI peptide, the cells did not display any responses, indicating that there was no non-specific or auto-reactive T cells present in the T cell line (Figure 1B). This indicates that a majority (>65–70%) of the population in the T cell line are specific to the SYVPSAEQI epitope.

Bottom Line: Presently, there is only one CD8(+) epitope, SYVPSAEQI, derived from the circumsporozoite protein of Plasmodium yoelii (PyCS), that mediates anti-malarial protection and is presented in the context of a K(d) molecule.We found that the adoptive transfer of a PyCS-specific CD8(+) T-cell line resulted in a significant inhibition of the parasite burden in the liver of Alb-K(d) Tg, as well as MHC-I-K(d) Tg mice, but not of C57BL/6 mice.These results indicate that the K(d) molecule expressed by hepatocytes is sufficient in mediating the anti-plasmodial activity of PyCS-specific CD8(+) T cells in vivo.

View Article: PubMed Central - PubMed

Affiliation: HIV and Malaria Vaccine Program, Aaron Diamond AIDS Research Center - The Rockefeller University New York, NY, USA.

ABSTRACT
Although CD8(+) T cells are shown to mediate the protective immunity against the liver stages of malaria parasites in mice, whether the direct presentation of malaria antigen by major histocompatibility complex (MHC) class I molecules expressed on the liver of infected host is required for anti-plasmodial activity of CD8(+) T cells is still unknown. Presently, there is only one CD8(+) epitope, SYVPSAEQI, derived from the circumsporozoite protein of Plasmodium yoelii (PyCS), that mediates anti-malarial protection and is presented in the context of a K(d) molecule. Therefore, to investigate the mode of anti-plasmodial activity of CD8+ T cells, we have previously generated C57BL/6 transgenic (Tg) mice, in which a K(d) molecule is expressed only on hepatocyte (Alb-K(d)) or dendritic cell (DC; CD11c-K(d)), by using albumin promoter or CD11c promoter, respectively. We have also generated MHC-I-K(d) Tg mice, which express the K(d) molecule under the MHC class I (MHC-I) promoter, as a positive control. From splenocytes collected from CD11c-K(d) Tg mice immunized with a synthetic peptide, SYVPSAEQI, which corresponds to the CD8(+) T-cell epitope of PyCS, emulsified in incomplete Freund's adjuvant , a PyCS-specific CD8(+) T-cell line was generated. This PyCS-specific CD8(+)T-cell line was then adoptively transferred into a cohort of either MHC-K(d) Tg or Alb-K(d) Tg mice listed above, as well as wild-type C57BL/6 mice. Then both transferred and non-transferred mice were challenged with live malaria parasites. We found that the adoptive transfer of a PyCS-specific CD8(+) T-cell line resulted in a significant inhibition of the parasite burden in the liver of Alb-K(d) Tg, as well as MHC-I-K(d) Tg mice, but not of C57BL/6 mice. These results indicate that the K(d) molecule expressed by hepatocytes is sufficient in mediating the anti-plasmodial activity of PyCS-specific CD8(+) T cells in vivo.

No MeSH data available.


Related in: MedlinePlus