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HLA-A2.1-restricted education and cytolytic activity of CD8(+) T lymphocytes from beta2 microglobulin (beta2m) HLA-A2.1 monochain transgenic H-2Db beta2m double knockout mice.

Pascolo S, Bervas N, Ure JM, Smith AG, Lemonnier FA, Pérarnau B - J. Exp. Med. (1997)

Bottom Line: Expression of this monochain restores a sizable peripheral CD8(+) T cell repertoire essentially educated on the transgenic human molecule.Interestingly, the CTL response to influenza A virus is mostly, if not exclusively, directed to the 58-66 matrix peptide which is the HLA-A2.1-restricted immunodominant epitope in humans.Such mice might constitute a versatile animal model for the study of HLA-A2.1-restricted CTL responses of vaccine interest.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Département SIDA-Rétrovirus, Unité d'Immunité Cellulaire Antivirale, 75724 Paris Cedex 15, France.

ABSTRACT
Three different HLA-A2.1 monochains were engineered in which either the human or mouse beta2-microglobulin (beta2m) is covalently linked to the NH2 terminus of the heavy chain by a 15- amino acid long peptide: HHH, entirely human, HHD, with the mouse H-2Db alpha3, transmembrane, and cytoplasmic domains, and MHD, homologous to HHD but linked to the mouse beta2mb. The cell surface expression and immunological capacities of the three monochains were compared with transfected cells, and the selected HHD construct was introduced by transgenesis in H-2Db-/- beta2m-/- double knockout mice. Expression of this monochain restores a sizable peripheral CD8(+) T cell repertoire essentially educated on the transgenic human molecule. Consequently, infected HHD, H-2Db-/- beta2m-/- mice generate only HLA-A2.1-restricted CD8(+) CTL responses against influenza A and vaccinia viruses. Interestingly, the CTL response to influenza A virus is mostly, if not exclusively, directed to the 58-66 matrix peptide which is the HLA-A2.1-restricted immunodominant epitope in humans. Such mice might constitute a versatile animal model for the study of HLA-A2.1-restricted CTL responses of vaccine interest.

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Phenotypical characterization of H-2Db−/−, β2m−/−, HHD  transgenic mice. Flow cytometric analyses were performed on spleen T  lymphocytes from H-2Db−/−, β2m−/−, HHD+ (left), or HHD− (right)  mice. (A) Expression of H-2Kb, H-2Db, and HHD molecules detected  with 20.8.4.S, B22.249.R.19, or B9.12.1 mAb, respectively and F(ab)′2  FITC-conjugated goat anti–mouse IgG, negative controls with no first  mAb. Results are expressed in fluorescence intensity (x-axis, log scale) and  relative cell number (y-axis). (B, top) Partial restoration of the peripheral  pool of CD8+ T lymphocytes. Double staining was performed with phycoerythrin-labeled anti-CD4 (x-axis, log scale) and biotinylated anti-CD8  detected with streptavidin–Perc-P (y-axis, log scale). (Bottom) Peripheral  CD8+ TCR repertoire. As illustrated for Vβ8.1.2.3, double staining was  performed with FITC-labeled anti-Vβ2 (B.20.6), -Vβ3 (KJ.25), -Vβ4  (KT.10.4), -Vβ5.1,.2 (MR.9.4), -Vβ6 (44.22), -Vβ7 (TR 130), -Vβ8.1,.2,.3  (F.23.1), -Vβ9 (MR.10.2), -Vβ10 (B.21.5), -Vβ11 (RR.3.15), -Vβ13  (MR.12.4), and -Vβ17 (KJ.23.1)–specific mAb (x-axis, log scale) and phycoerythrin-labeled anti-CD8 mAb (y-axis, log scale).
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Figure 7: Phenotypical characterization of H-2Db−/−, β2m−/−, HHD transgenic mice. Flow cytometric analyses were performed on spleen T lymphocytes from H-2Db−/−, β2m−/−, HHD+ (left), or HHD− (right) mice. (A) Expression of H-2Kb, H-2Db, and HHD molecules detected with 20.8.4.S, B22.249.R.19, or B9.12.1 mAb, respectively and F(ab)′2 FITC-conjugated goat anti–mouse IgG, negative controls with no first mAb. Results are expressed in fluorescence intensity (x-axis, log scale) and relative cell number (y-axis). (B, top) Partial restoration of the peripheral pool of CD8+ T lymphocytes. Double staining was performed with phycoerythrin-labeled anti-CD4 (x-axis, log scale) and biotinylated anti-CD8 detected with streptavidin–Perc-P (y-axis, log scale). (Bottom) Peripheral CD8+ TCR repertoire. As illustrated for Vβ8.1.2.3, double staining was performed with FITC-labeled anti-Vβ2 (B.20.6), -Vβ3 (KJ.25), -Vβ4 (KT.10.4), -Vβ5.1,.2 (MR.9.4), -Vβ6 (44.22), -Vβ7 (TR 130), -Vβ8.1,.2,.3 (F.23.1), -Vβ9 (MR.10.2), -Vβ10 (B.21.5), -Vβ11 (RR.3.15), -Vβ13 (MR.12.4), and -Vβ17 (KJ.23.1)–specific mAb (x-axis, log scale) and phycoerythrin-labeled anti-CD8 mAb (y-axis, log scale).

Mentions: Weak (compared to endogenous H-2 class I molecules in normal mice), but significant, expression of the transgenic HHD molecules on unactivated peripheral T lymphocytes was documented by indirect immunofluorescence and FACS® analysis. Under similar conditions, no H-2Kb and H-2Db molecules could be detected (Fig. 7 A). This expression of HHD monochains partially restores the peripheral pool of CD8+ T lymphocytes (5.5% of the T lymphocytes instead of 20–30% in normal mice). More importantly, testing the 10 available anti-Vβ mAb, it appeared, as illustrated in Fig. 7 B for Vβ8, that these CD8+ T lymphocytes exhibited a diversified TCR repertoire. By comparison with H-2Db−/− β2m−/− double knockout mice, which are almost completely deprived of peripheral CD8+ T lymphocytes, these results suggest that HHD monochains promote CD8+ T cell positive education in the thymus.


HLA-A2.1-restricted education and cytolytic activity of CD8(+) T lymphocytes from beta2 microglobulin (beta2m) HLA-A2.1 monochain transgenic H-2Db beta2m double knockout mice.

Pascolo S, Bervas N, Ure JM, Smith AG, Lemonnier FA, Pérarnau B - J. Exp. Med. (1997)

Phenotypical characterization of H-2Db−/−, β2m−/−, HHD  transgenic mice. Flow cytometric analyses were performed on spleen T  lymphocytes from H-2Db−/−, β2m−/−, HHD+ (left), or HHD− (right)  mice. (A) Expression of H-2Kb, H-2Db, and HHD molecules detected  with 20.8.4.S, B22.249.R.19, or B9.12.1 mAb, respectively and F(ab)′2  FITC-conjugated goat anti–mouse IgG, negative controls with no first  mAb. Results are expressed in fluorescence intensity (x-axis, log scale) and  relative cell number (y-axis). (B, top) Partial restoration of the peripheral  pool of CD8+ T lymphocytes. Double staining was performed with phycoerythrin-labeled anti-CD4 (x-axis, log scale) and biotinylated anti-CD8  detected with streptavidin–Perc-P (y-axis, log scale). (Bottom) Peripheral  CD8+ TCR repertoire. As illustrated for Vβ8.1.2.3, double staining was  performed with FITC-labeled anti-Vβ2 (B.20.6), -Vβ3 (KJ.25), -Vβ4  (KT.10.4), -Vβ5.1,.2 (MR.9.4), -Vβ6 (44.22), -Vβ7 (TR 130), -Vβ8.1,.2,.3  (F.23.1), -Vβ9 (MR.10.2), -Vβ10 (B.21.5), -Vβ11 (RR.3.15), -Vβ13  (MR.12.4), and -Vβ17 (KJ.23.1)–specific mAb (x-axis, log scale) and phycoerythrin-labeled anti-CD8 mAb (y-axis, log scale).
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Related In: Results  -  Collection

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Figure 7: Phenotypical characterization of H-2Db−/−, β2m−/−, HHD transgenic mice. Flow cytometric analyses were performed on spleen T lymphocytes from H-2Db−/−, β2m−/−, HHD+ (left), or HHD− (right) mice. (A) Expression of H-2Kb, H-2Db, and HHD molecules detected with 20.8.4.S, B22.249.R.19, or B9.12.1 mAb, respectively and F(ab)′2 FITC-conjugated goat anti–mouse IgG, negative controls with no first mAb. Results are expressed in fluorescence intensity (x-axis, log scale) and relative cell number (y-axis). (B, top) Partial restoration of the peripheral pool of CD8+ T lymphocytes. Double staining was performed with phycoerythrin-labeled anti-CD4 (x-axis, log scale) and biotinylated anti-CD8 detected with streptavidin–Perc-P (y-axis, log scale). (Bottom) Peripheral CD8+ TCR repertoire. As illustrated for Vβ8.1.2.3, double staining was performed with FITC-labeled anti-Vβ2 (B.20.6), -Vβ3 (KJ.25), -Vβ4 (KT.10.4), -Vβ5.1,.2 (MR.9.4), -Vβ6 (44.22), -Vβ7 (TR 130), -Vβ8.1,.2,.3 (F.23.1), -Vβ9 (MR.10.2), -Vβ10 (B.21.5), -Vβ11 (RR.3.15), -Vβ13 (MR.12.4), and -Vβ17 (KJ.23.1)–specific mAb (x-axis, log scale) and phycoerythrin-labeled anti-CD8 mAb (y-axis, log scale).
Mentions: Weak (compared to endogenous H-2 class I molecules in normal mice), but significant, expression of the transgenic HHD molecules on unactivated peripheral T lymphocytes was documented by indirect immunofluorescence and FACS® analysis. Under similar conditions, no H-2Kb and H-2Db molecules could be detected (Fig. 7 A). This expression of HHD monochains partially restores the peripheral pool of CD8+ T lymphocytes (5.5% of the T lymphocytes instead of 20–30% in normal mice). More importantly, testing the 10 available anti-Vβ mAb, it appeared, as illustrated in Fig. 7 B for Vβ8, that these CD8+ T lymphocytes exhibited a diversified TCR repertoire. By comparison with H-2Db−/− β2m−/− double knockout mice, which are almost completely deprived of peripheral CD8+ T lymphocytes, these results suggest that HHD monochains promote CD8+ T cell positive education in the thymus.

Bottom Line: Expression of this monochain restores a sizable peripheral CD8(+) T cell repertoire essentially educated on the transgenic human molecule.Interestingly, the CTL response to influenza A virus is mostly, if not exclusively, directed to the 58-66 matrix peptide which is the HLA-A2.1-restricted immunodominant epitope in humans.Such mice might constitute a versatile animal model for the study of HLA-A2.1-restricted CTL responses of vaccine interest.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Département SIDA-Rétrovirus, Unité d'Immunité Cellulaire Antivirale, 75724 Paris Cedex 15, France.

ABSTRACT
Three different HLA-A2.1 monochains were engineered in which either the human or mouse beta2-microglobulin (beta2m) is covalently linked to the NH2 terminus of the heavy chain by a 15- amino acid long peptide: HHH, entirely human, HHD, with the mouse H-2Db alpha3, transmembrane, and cytoplasmic domains, and MHD, homologous to HHD but linked to the mouse beta2mb. The cell surface expression and immunological capacities of the three monochains were compared with transfected cells, and the selected HHD construct was introduced by transgenesis in H-2Db-/- beta2m-/- double knockout mice. Expression of this monochain restores a sizable peripheral CD8(+) T cell repertoire essentially educated on the transgenic human molecule. Consequently, infected HHD, H-2Db-/- beta2m-/- mice generate only HLA-A2.1-restricted CD8(+) CTL responses against influenza A and vaccinia viruses. Interestingly, the CTL response to influenza A virus is mostly, if not exclusively, directed to the 58-66 matrix peptide which is the HLA-A2.1-restricted immunodominant epitope in humans. Such mice might constitute a versatile animal model for the study of HLA-A2.1-restricted CTL responses of vaccine interest.

Show MeSH
Related in: MedlinePlus