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Lineage relationships and differentiation of natural killer (NK) T cells: intrathymic selection and interleukin (IL)-4 production in the absence of NKR-P1 and Ly49 molecules.

Lantz O, Sharara LI, Tilloy F, Andersson A, DiSanto JP - J. Exp. Med. (1997)

Bottom Line: In this report, we have assessed the lineage relationships and cytokine dependency of natural killer (NK) T cells compared with mainstream TCR-alphabeta T cells and NK cells.However, gamma c-deficient NK thymocytes fail to coexpress the NK-associated markers NKR-P1 or Ly49, yet retain characteristic expression of the cytokine receptors interleukin (IL)-7R alpha and IL-2Rbeta.These results suggest a stepwise pattern of differentiation for thymically derived NK T cells: primary selection via their invariant TCR to confer the IL-4-producing phenotype, followed by acquisition of NK-associated markers and maturation/export to the periphery.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Medicale U267, Hôpital Paul Brousse, Villejuif, France.

ABSTRACT
In this report, we have assessed the lineage relationships and cytokine dependency of natural killer (NK) T cells compared with mainstream TCR-alphabeta T cells and NK cells. For this purpose, we studied common gamma chain (gamma c)-deficient mice, which demonstrate a selective defect in CD3- NK cell development relative to conventional TCR-alphabeta T cells. NK thymocytes differentiate in gamma c- mice as shown by the normal percentage of TCR Vbeta8+ CD4-CD8- cells and the normal quantity of thymic Va14-Jalpha281 mRNA that characterize the NK T repertoire. However, gamma c-deficient NK thymocytes fail to coexpress the NK-associated markers NKR-P1 or Ly49, yet retain characteristic expression of the cytokine receptors interleukin (IL)-7R alpha and IL-2Rbeta. Despite these phenotypic abnormalities, gamma c- NK thymocytes could produce normal amounts of IL-4. These results define a maturational progression of NK thymocyte differentiation where intrathymic selection and IL-4-producing capacity can be clearly dissociated from the acquisition of the NK phenotype. Moreover, these data suggest a closer ontogenic relationship of NK T cells to TCR-alphabeta T cells than to NK cells with respect to cytokine dependency. We also failed to detect peripheral NK T cells in these mice, demonstrating that gamma c-dependent interactions are required for export and/or survival of NK T cells from the thymus. These results suggest a stepwise pattern of differentiation for thymically derived NK T cells: primary selection via their invariant TCR to confer the IL-4-producing phenotype, followed by acquisition of NK-associated markers and maturation/export to the periphery.

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Vα14–Jα281 invariant α chain is absent from the liver of γc−  mice. Duplicates samples of 3 × 105 liver lymphocytes were obtained  from the indicated mice and processed as indicated in Fig. 1. It should be  noticed that the amount of Cα is lower in the γc+ preps. The differences  in Vα14–Jα281 amounts between γc+ and β2m−/− samples is at least  100-fold (5+3 cycles) and between γc+ and γc− at least 200-fold (7+2  cycles). Representative of three independent experiments.
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Figure 4: Vα14–Jα281 invariant α chain is absent from the liver of γc− mice. Duplicates samples of 3 × 105 liver lymphocytes were obtained from the indicated mice and processed as indicated in Fig. 1. It should be noticed that the amount of Cα is lower in the γc+ preps. The differences in Vα14–Jα281 amounts between γc+ and β2m−/− samples is at least 100-fold (5+3 cycles) and between γc+ and γc− at least 200-fold (7+2 cycles). Representative of three independent experiments.

Mentions: Next, we examined whether γc− NK thymocytes, despite their phenotypic abnormalities, would be able to attain their preferential localizations in the periphery. NK T cells normally comprise a small percentage of the lymphocytes present in the spleen and lymph nodes (1–3); however, these cells are abundant in the liver (20, 30). To quantitate peripheral NK T cells, lymphocytes from γc+, γc−, or β2m−/− mice were isolated from the liver and spleen and the amount of Vα14–Jα281 mRNA was determined. NK T cells were clearly present in the liver and spleens of γc+ mice (Fig. 4; data not shown) as evidenced by the presence of Vα14– Jα281+ mRNA. In contrast, levels of Vα14–Jα281+ mRNA from γc− liver and spleen preparations were at or below that of β2m−/− mice (which lack NK T cells) and well below that of γc+ controls (at least 200-fold less). Polyclonal sequencing of Vα14–Jα281 amplicons from γc+, γc−, or β2m−/− samples showed an invariant sequence only in the γc+ samples (data not shown). Flow cytometric analyses confirmed the presence of NK1.1+ TCR-αβint cells in intrahepatic lymphocytes from γc+ mice, which were not detected in preparations from γc− mice (Fig. 5). Lastly, in vivo administration of anti-CD3 antibodies stimulated IL-4 release from cultured γc+ splenocytes, whereas no IL-4 production could be detected in splenocyte cultures from γc− mice (Table 2). Taken together, these results demonstrate an absence of NK T cells in the liver and spleen of γc− mice.


Lineage relationships and differentiation of natural killer (NK) T cells: intrathymic selection and interleukin (IL)-4 production in the absence of NKR-P1 and Ly49 molecules.

Lantz O, Sharara LI, Tilloy F, Andersson A, DiSanto JP - J. Exp. Med. (1997)

Vα14–Jα281 invariant α chain is absent from the liver of γc−  mice. Duplicates samples of 3 × 105 liver lymphocytes were obtained  from the indicated mice and processed as indicated in Fig. 1. It should be  noticed that the amount of Cα is lower in the γc+ preps. The differences  in Vα14–Jα281 amounts between γc+ and β2m−/− samples is at least  100-fold (5+3 cycles) and between γc+ and γc− at least 200-fold (7+2  cycles). Representative of three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196284&req=5

Figure 4: Vα14–Jα281 invariant α chain is absent from the liver of γc− mice. Duplicates samples of 3 × 105 liver lymphocytes were obtained from the indicated mice and processed as indicated in Fig. 1. It should be noticed that the amount of Cα is lower in the γc+ preps. The differences in Vα14–Jα281 amounts between γc+ and β2m−/− samples is at least 100-fold (5+3 cycles) and between γc+ and γc− at least 200-fold (7+2 cycles). Representative of three independent experiments.
Mentions: Next, we examined whether γc− NK thymocytes, despite their phenotypic abnormalities, would be able to attain their preferential localizations in the periphery. NK T cells normally comprise a small percentage of the lymphocytes present in the spleen and lymph nodes (1–3); however, these cells are abundant in the liver (20, 30). To quantitate peripheral NK T cells, lymphocytes from γc+, γc−, or β2m−/− mice were isolated from the liver and spleen and the amount of Vα14–Jα281 mRNA was determined. NK T cells were clearly present in the liver and spleens of γc+ mice (Fig. 4; data not shown) as evidenced by the presence of Vα14– Jα281+ mRNA. In contrast, levels of Vα14–Jα281+ mRNA from γc− liver and spleen preparations were at or below that of β2m−/− mice (which lack NK T cells) and well below that of γc+ controls (at least 200-fold less). Polyclonal sequencing of Vα14–Jα281 amplicons from γc+, γc−, or β2m−/− samples showed an invariant sequence only in the γc+ samples (data not shown). Flow cytometric analyses confirmed the presence of NK1.1+ TCR-αβint cells in intrahepatic lymphocytes from γc+ mice, which were not detected in preparations from γc− mice (Fig. 5). Lastly, in vivo administration of anti-CD3 antibodies stimulated IL-4 release from cultured γc+ splenocytes, whereas no IL-4 production could be detected in splenocyte cultures from γc− mice (Table 2). Taken together, these results demonstrate an absence of NK T cells in the liver and spleen of γc− mice.

Bottom Line: In this report, we have assessed the lineage relationships and cytokine dependency of natural killer (NK) T cells compared with mainstream TCR-alphabeta T cells and NK cells.However, gamma c-deficient NK thymocytes fail to coexpress the NK-associated markers NKR-P1 or Ly49, yet retain characteristic expression of the cytokine receptors interleukin (IL)-7R alpha and IL-2Rbeta.These results suggest a stepwise pattern of differentiation for thymically derived NK T cells: primary selection via their invariant TCR to confer the IL-4-producing phenotype, followed by acquisition of NK-associated markers and maturation/export to the periphery.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Medicale U267, Hôpital Paul Brousse, Villejuif, France.

ABSTRACT
In this report, we have assessed the lineage relationships and cytokine dependency of natural killer (NK) T cells compared with mainstream TCR-alphabeta T cells and NK cells. For this purpose, we studied common gamma chain (gamma c)-deficient mice, which demonstrate a selective defect in CD3- NK cell development relative to conventional TCR-alphabeta T cells. NK thymocytes differentiate in gamma c- mice as shown by the normal percentage of TCR Vbeta8+ CD4-CD8- cells and the normal quantity of thymic Va14-Jalpha281 mRNA that characterize the NK T repertoire. However, gamma c-deficient NK thymocytes fail to coexpress the NK-associated markers NKR-P1 or Ly49, yet retain characteristic expression of the cytokine receptors interleukin (IL)-7R alpha and IL-2Rbeta. Despite these phenotypic abnormalities, gamma c- NK thymocytes could produce normal amounts of IL-4. These results define a maturational progression of NK thymocyte differentiation where intrathymic selection and IL-4-producing capacity can be clearly dissociated from the acquisition of the NK phenotype. Moreover, these data suggest a closer ontogenic relationship of NK T cells to TCR-alphabeta T cells than to NK cells with respect to cytokine dependency. We also failed to detect peripheral NK T cells in these mice, demonstrating that gamma c-dependent interactions are required for export and/or survival of NK T cells from the thymus. These results suggest a stepwise pattern of differentiation for thymically derived NK T cells: primary selection via their invariant TCR to confer the IL-4-producing phenotype, followed by acquisition of NK-associated markers and maturation/export to the periphery.

Show MeSH
Related in: MedlinePlus