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

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.

Figure 1.

Human CD127⁺ LTi-like cells are precursors of CD56⁺CD127⁺ cells and have similar phenotype to NK22 cells. (A) Lin⁻, CD56⁺ cNK, CD56⁺CD127⁺, or CD127⁺ LTi-like cells, were sorted from tonsil and were co-cultured with human MSCs for 4 d with 10 ng/ml IL-7. Expression of ICAM1 and VCAM1 on MSCs was quantified by flow cytometry. (B) Expression of the indicated molecules was quantified using flow cytometry on Lin⁻ tonsil cells. (C) Flow cytometry–sorted CD127⁺ LTi cells were labeled with CFSE and stimulated with IL-7, IL-2, or IL-15 for 6 d, followed by flow cytometry to assess CD56 expression. One donor out of at least two analyzed is shown in A, B, and C.

Figure 2.

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.

Figure 3.

Heterogeneous cytokine production from expanded and clonal CD127⁺ LTi-like cells. (A) Cell lines were stimulated with PMA and ionomycin for 24 h, supernatants were collected, and cytokine production was analyzed by Luminex assays and ELISA. (B) Intracellular cytokine analysis of expanded cNK and CD127⁺LTi-like cell lines, stimulated for 6 h with PMA and ionomycin. (C and D) Intracellular cytokine analysis of LTi cell clones, stimulated for 6 h with PMA and ionomycin. In A, the mean value of three donors is plotted. B shows a single donor representative of at least three donors. In C, each dot represents a separate clone, with two donors shown. In D, three representative clones are shown from each donor. Error bars in A and C show SEM. Horizontal bars in C show means.

Figure 4.

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.

Figure 5.

CD117⁺CD161⁺CD127^- iNK cells contain cNK precursors. (A) Lin⁻CD117⁺CD161⁺CD127⁻ and CD117⁺CD161⁺CD127⁺ cells were sorted from human tonsils. (B) Isolated populations were expanded ex vivo, and expression of indicated molecules was assessed by flow cytometry. (C) RORC, IL-22, and IFN-γ mRNA after ex vivo expansion, normalized to 18S expression. A single donor representative of two is shown in A and B, and C represents the mean and SEM of at least two donors.