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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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Cultured CD56+CD127+ and CD127+ LTi-like cells maintain high RORC expression. Flow cytometry–sorted tonsil Lin− cNK, CD117+CD56+CD127+, and CD117+CD127+ LTi-like cells were expanded ex vivo using a feeder cell mixture. (A) After expansion, RORC mRNA normalized to 18S expression. The top represents the first expansion (F1), whereas the bottom follows RORC expression in a single donor over three rounds of expansion. (B) Flow cytometry of the indicated molecules on expanded cell lines after one (F1) or three (F3) rounds of expansion. (C) Single cell clones of CD117+CD127+ LTi-like cells were generated, and RORC mRNA of clones from two donors is shown, normalized to 18S expression. Bulk cell lines from the same donors are shown in parallel. (D) Correlation between AHR and RORC mRNA from LTi-like cell clones. (E) Flow cytometry of the indicated molecules on LTi-like cell clones. Each dot in A, C, and D represents a single clone or bulk cell line. In B, a single donor representative of five is shown. In E, three representative clones are shown. The mean (horizontal bars) and SEM (error bars) are shown in A and C.
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fig2: Cultured CD56+CD127+ and CD127+ LTi-like cells maintain high RORC expression. Flow cytometry–sorted tonsil Lin− cNK, CD117+CD56+CD127+, and CD117+CD127+ LTi-like cells were expanded ex vivo using a feeder cell mixture. (A) After expansion, RORC mRNA normalized to 18S expression. The top represents the first expansion (F1), whereas the bottom follows RORC expression in a single donor over three rounds of expansion. (B) Flow cytometry of the indicated molecules on expanded cell lines after one (F1) or three (F3) rounds of expansion. (C) Single cell clones of CD117+CD127+ LTi-like cells were generated, and RORC mRNA of clones from two donors is shown, normalized to 18S expression. Bulk cell lines from the same donors are shown in parallel. (D) Correlation between AHR and RORC mRNA from LTi-like cell clones. (E) Flow cytometry of the indicated molecules on LTi-like cell clones. Each dot in A, C, and D represents a single clone or bulk cell line. In B, a single donor representative of five is shown. In E, three representative clones are shown. The mean (horizontal bars) and SEM (error bars) are shown in A and C.

Mentions: Immature stage 3 NK cells have been shown to develop into cNK cells (Freud et al., 2006). Because considerable phenotypic similarities exist between LTi-like cells and stage 3 iNK cells, we examined whether CD127+ LTi-like cells can convert into cNK cells. Using a rigorous sorting strategy, we isolated CD56+CD117− cNK cells, Lin−CD56+CD117+CD127+ cells, and Lin−CD56−CD117+CD127+ LTi-like cells from human tonsils. The use of CD117, in conjunction with CD127 as selecting markers, combined with the rigorous exclusion of Lin+ and/or TCR+ cells, was crucial for preventing contamination with T cells. These sorted populations were expanded ex vivo using a feeder mixture consisting of irradiated allogeneic PBMCs, JY EBV-transformed lymphoblastoid cells, PHA, and IL-2, which was previously used to expand cNK cells (Phillips et al., 1991). Under these conditions, CD127+ LTi-like cells expanded 30–200-fold, and after resting they could be successfully restimulated and further expanded. Importantly, expanded LTi-like cell lines maintained expression of RORC messenger RNA (mRNA), as did CD56+CD127+ cells, whereas CD56+ cNK cells remained negative (Fig. 2 A). At the end of the first feeder cycle, LTi-like cell lines were ∼30–60% CD56+ (Fig. 2 B). After subsequent feeder cycles, CD56 expression increased until ∼80–100% of the cells were CD56+. After expansion, all populations expressed similar levels of NKp46. CD127 was no longer detectable by flow cytometry; however, LTi-like cell lines continued to express CD127 message (Fig. S1) and to respond to IL-7 (unpublished data). The down-regulation of CD127 is not unexpected, as it is well known that T cells lose CD127 expression after activation and expansion (Xue et al., 2002). Expanded cNK cells expressed CD56, but in contrast to cell lines derived from CD127+CD56− and CD127+CD56+ cells, they lacked CD117.


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)

Cultured CD56+CD127+ and CD127+ LTi-like cells maintain high RORC expression. Flow cytometry–sorted tonsil Lin− cNK, CD117+CD56+CD127+, and CD117+CD127+ LTi-like cells were expanded ex vivo using a feeder cell mixture. (A) After expansion, RORC mRNA normalized to 18S expression. The top represents the first expansion (F1), whereas the bottom follows RORC expression in a single donor over three rounds of expansion. (B) Flow cytometry of the indicated molecules on expanded cell lines after one (F1) or three (F3) rounds of expansion. (C) Single cell clones of CD117+CD127+ LTi-like cells were generated, and RORC mRNA of clones from two donors is shown, normalized to 18S expression. Bulk cell lines from the same donors are shown in parallel. (D) Correlation between AHR and RORC mRNA from LTi-like cell clones. (E) Flow cytometry of the indicated molecules on LTi-like cell clones. Each dot in A, C, and D represents a single clone or bulk cell line. In B, a single donor representative of five is shown. In E, three representative clones are shown. The mean (horizontal bars) and SEM (error bars) are shown in A and C.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig2: Cultured CD56+CD127+ and CD127+ LTi-like cells maintain high RORC expression. Flow cytometry–sorted tonsil Lin− cNK, CD117+CD56+CD127+, and CD117+CD127+ LTi-like cells were expanded ex vivo using a feeder cell mixture. (A) After expansion, RORC mRNA normalized to 18S expression. The top represents the first expansion (F1), whereas the bottom follows RORC expression in a single donor over three rounds of expansion. (B) Flow cytometry of the indicated molecules on expanded cell lines after one (F1) or three (F3) rounds of expansion. (C) Single cell clones of CD117+CD127+ LTi-like cells were generated, and RORC mRNA of clones from two donors is shown, normalized to 18S expression. Bulk cell lines from the same donors are shown in parallel. (D) Correlation between AHR and RORC mRNA from LTi-like cell clones. (E) Flow cytometry of the indicated molecules on LTi-like cell clones. Each dot in A, C, and D represents a single clone or bulk cell line. In B, a single donor representative of five is shown. In E, three representative clones are shown. The mean (horizontal bars) and SEM (error bars) are shown in A and C.
Mentions: Immature stage 3 NK cells have been shown to develop into cNK cells (Freud et al., 2006). Because considerable phenotypic similarities exist between LTi-like cells and stage 3 iNK cells, we examined whether CD127+ LTi-like cells can convert into cNK cells. Using a rigorous sorting strategy, we isolated CD56+CD117− cNK cells, Lin−CD56+CD117+CD127+ cells, and Lin−CD56−CD117+CD127+ LTi-like cells from human tonsils. The use of CD117, in conjunction with CD127 as selecting markers, combined with the rigorous exclusion of Lin+ and/or TCR+ cells, was crucial for preventing contamination with T cells. These sorted populations were expanded ex vivo using a feeder mixture consisting of irradiated allogeneic PBMCs, JY EBV-transformed lymphoblastoid cells, PHA, and IL-2, which was previously used to expand cNK cells (Phillips et al., 1991). Under these conditions, CD127+ LTi-like cells expanded 30–200-fold, and after resting they could be successfully restimulated and further expanded. Importantly, expanded LTi-like cell lines maintained expression of RORC messenger RNA (mRNA), as did CD56+CD127+ cells, whereas CD56+ cNK cells remained negative (Fig. 2 A). At the end of the first feeder cycle, LTi-like cell lines were ∼30–60% CD56+ (Fig. 2 B). After subsequent feeder cycles, CD56 expression increased until ∼80–100% of the cells were CD56+. After expansion, all populations expressed similar levels of NKp46. CD127 was no longer detectable by flow cytometry; however, LTi-like cell lines continued to express CD127 message (Fig. S1) and to respond to IL-7 (unpublished data). The down-regulation of CD127 is not unexpected, as it is well known that T cells lose CD127 expression after activation and expansion (Xue et al., 2002). Expanded cNK cells expressed CD56, but in contrast to cell lines derived from CD127+CD56− and CD127+CD56+ cells, they lacked CD117.

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.

Show MeSH
Related in: MedlinePlus