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Human NKp44+IL-22+ cells and LTi-like cells constitute a stable RORC+ lineage distinct from conventional natural killer cells.

Crellin NK, Trifari S, Kaplan CD, Cupedo T, Spits H - J. Exp. Med. (2010)

Bottom Line: Clonal analysis reveals heterogeneity of cytokine production within the CD127(+) LTi-like population.Furthermore, we identify within the tonsil a cNK precursor population that is characterized as Lin(-)CD117(+)CD161(+)CD127(-) cells.Overall, we propose that CD127(+)RORC(+) cells, although they share some characteristics with cNK cells, represent a functionally and developmentally distinct lineage.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Genentech, South San Francisco, CA 94080, USA.

ABSTRACT
Lymphoid tissue inducer (LTi) cells are required for lymph node formation during fetal development, and recent evidence implies a role in mucosal immunity in the adult. LTi cells share some phenotypic features of conventional natural killer (NK; cNK) cells; however, little is known to date about the relationship between these two cell types. We show that lineage(-) (Lin(-)) CD127(+)RORC(+) LTi-like cells in human tonsil are precursors to CD56(+)CD127(+)RORC(+)NKp46(+) cells, which together comprise a stable RORC(+) lineage. We find that LTi-like cells and their CD56(+) progeny can be expanded and cloned ex vivo without loss of function and without conversion into cNK cells. Clonal analysis reveals heterogeneity of cytokine production within the CD127(+) LTi-like population. Furthermore, we identify within the tonsil a cNK precursor population that is characterized as Lin(-)CD117(+)CD161(+)CD127(-) cells. Overall, we propose that CD127(+)RORC(+) cells, although they share some characteristics with cNK cells, represent a functionally and developmentally distinct lineage.

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After ex vivo expansion, LTi cell lines have low cytotoxic activity. (A) Flow cytometry was performed for intracellular perforin, granzyme B, and cell surface KIR expression on cNK cell lines and CD127+ LTi-like cell lines expanded ex vivo. (B) Geometric mean fluorescence intensity of intracellular granzyme B or perforin staining is shown for cNK or CD127+ LTi-like cell lines and LTi-like clones after ex vivo expansion. (C) K562 cells were labeled with a fluorescent dye and co-cultured with cNK or CD127+ LTi-like cell lines or LTi-like clones at the indicated ratios for 5 h, and cytotoxicity was determined by propidium iodide staining. Unlabeled K562 cells were added at the indicated ratios to assess nonspecific cell death (control). One of four donors analyzed is shown in A. Each dot represents a separate donor or clone in B. The mean of cell lines from two donors and three clones from a single donor are shown in C. Horizontal bars in B show the means. Error bars in B and C show SEM.
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fig4: After ex vivo expansion, LTi cell lines have low cytotoxic activity. (A) Flow cytometry was performed for intracellular perforin, granzyme B, and cell surface KIR expression on cNK cell lines and CD127+ LTi-like cell lines expanded ex vivo. (B) Geometric mean fluorescence intensity of intracellular granzyme B or perforin staining is shown for cNK or CD127+ LTi-like cell lines and LTi-like clones after ex vivo expansion. (C) K562 cells were labeled with a fluorescent dye and co-cultured with cNK or CD127+ LTi-like cell lines or LTi-like clones at the indicated ratios for 5 h, and cytotoxicity was determined by propidium iodide staining. Unlabeled K562 cells were added at the indicated ratios to assess nonspecific cell death (control). One of four donors analyzed is shown in A. Each dot represents a separate donor or clone in B. The mean of cell lines from two donors and three clones from a single donor are shown in C. Horizontal bars in B show the means. Error bars in B and C show SEM.

Mentions: We, and others, have documented that freshly isolated LTi-like cells from the tonsil, like their fetal counterparts, are negative for granzyme and perforin (Cella et al., 2009; Cupedo et al., 2009; Sanos et al., 2009). Because CD56+ LTi-like cell lines were capable of producing IFN-γ (Fig. 3), we assessed whether they acquired cytotoxic activity. After expansion, LTi-like cell lines and clones expressed perforin, but at much lower levels compared with cNK cell lines, and did not acquire expression of killer cell immunoglobulin-like receptors (KIRs; Fig. 4, A and B; and not depicted). In contrast to LTi-like cell clones, LTi-like cell lines did express some granzyme B but at much lower than cNK cell lines (Fig. 4, A and B). In a cytotoxic assay with K562 cells as targets, cultured LTi-like cells achieved 50% lysis at an effector/target ratio of 19:1, which is ∼13-fold higher than that required for cNK cell lines (Fig. 4 C), suggesting that in vitro–cultured LTi-like cells acquired a moderate cytolytic activity. However, this activity was much lower than that of cNK cells. The clones derived from LTi-like cells did not display any cytotoxicity in this assay. Collectively, our data support the notion that LTi and their CD127+CD56+ progeny represent a lineage whose developmental pathway is distinct from cNK cells.


Human NKp44+IL-22+ cells and LTi-like cells constitute a stable RORC+ lineage distinct from conventional natural killer cells.

Crellin NK, Trifari S, Kaplan CD, Cupedo T, Spits H - J. Exp. Med. (2010)

After ex vivo expansion, LTi cell lines have low cytotoxic activity. (A) Flow cytometry was performed for intracellular perforin, granzyme B, and cell surface KIR expression on cNK cell lines and CD127+ LTi-like cell lines expanded ex vivo. (B) Geometric mean fluorescence intensity of intracellular granzyme B or perforin staining is shown for cNK or CD127+ LTi-like cell lines and LTi-like clones after ex vivo expansion. (C) K562 cells were labeled with a fluorescent dye and co-cultured with cNK or CD127+ LTi-like cell lines or LTi-like clones at the indicated ratios for 5 h, and cytotoxicity was determined by propidium iodide staining. Unlabeled K562 cells were added at the indicated ratios to assess nonspecific cell death (control). One of four donors analyzed is shown in A. Each dot represents a separate donor or clone in B. The mean of cell lines from two donors and three clones from a single donor are shown in C. Horizontal bars in B show the means. Error bars in B and C show SEM.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig4: After ex vivo expansion, LTi cell lines have low cytotoxic activity. (A) Flow cytometry was performed for intracellular perforin, granzyme B, and cell surface KIR expression on cNK cell lines and CD127+ LTi-like cell lines expanded ex vivo. (B) Geometric mean fluorescence intensity of intracellular granzyme B or perforin staining is shown for cNK or CD127+ LTi-like cell lines and LTi-like clones after ex vivo expansion. (C) K562 cells were labeled with a fluorescent dye and co-cultured with cNK or CD127+ LTi-like cell lines or LTi-like clones at the indicated ratios for 5 h, and cytotoxicity was determined by propidium iodide staining. Unlabeled K562 cells were added at the indicated ratios to assess nonspecific cell death (control). One of four donors analyzed is shown in A. Each dot represents a separate donor or clone in B. The mean of cell lines from two donors and three clones from a single donor are shown in C. Horizontal bars in B show the means. Error bars in B and C show SEM.
Mentions: We, and others, have documented that freshly isolated LTi-like cells from the tonsil, like their fetal counterparts, are negative for granzyme and perforin (Cella et al., 2009; Cupedo et al., 2009; Sanos et al., 2009). Because CD56+ LTi-like cell lines were capable of producing IFN-γ (Fig. 3), we assessed whether they acquired cytotoxic activity. After expansion, LTi-like cell lines and clones expressed perforin, but at much lower levels compared with cNK cell lines, and did not acquire expression of killer cell immunoglobulin-like receptors (KIRs; Fig. 4, A and B; and not depicted). In contrast to LTi-like cell clones, LTi-like cell lines did express some granzyme B but at much lower than cNK cell lines (Fig. 4, A and B). In a cytotoxic assay with K562 cells as targets, cultured LTi-like cells achieved 50% lysis at an effector/target ratio of 19:1, which is ∼13-fold higher than that required for cNK cell lines (Fig. 4 C), suggesting that in vitro–cultured LTi-like cells acquired a moderate cytolytic activity. However, this activity was much lower than that of cNK cells. The clones derived from LTi-like cells did not display any cytotoxicity in this assay. Collectively, our data support the notion that LTi and their CD127+CD56+ progeny represent a lineage whose developmental pathway is distinct from cNK cells.

Bottom Line: Clonal analysis reveals heterogeneity of cytokine production within the CD127(+) LTi-like population.Furthermore, we identify within the tonsil a cNK precursor population that is characterized as Lin(-)CD117(+)CD161(+)CD127(-) cells.Overall, we propose that CD127(+)RORC(+) cells, although they share some characteristics with cNK cells, represent a functionally and developmentally distinct lineage.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Genentech, South San Francisco, CA 94080, USA.

ABSTRACT
Lymphoid tissue inducer (LTi) cells are required for lymph node formation during fetal development, and recent evidence implies a role in mucosal immunity in the adult. LTi cells share some phenotypic features of conventional natural killer (NK; cNK) cells; however, little is known to date about the relationship between these two cell types. We show that lineage(-) (Lin(-)) CD127(+)RORC(+) LTi-like cells in human tonsil are precursors to CD56(+)CD127(+)RORC(+)NKp46(+) cells, which together comprise a stable RORC(+) lineage. We find that LTi-like cells and their CD56(+) progeny can be expanded and cloned ex vivo without loss of function and without conversion into cNK cells. Clonal analysis reveals heterogeneity of cytokine production within the CD127(+) LTi-like population. Furthermore, we identify within the tonsil a cNK precursor population that is characterized as Lin(-)CD117(+)CD161(+)CD127(-) cells. Overall, we propose that CD127(+)RORC(+) cells, although they share some characteristics with cNK cells, represent a functionally and developmentally distinct lineage.

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