NK‐like activity against allogeneic tumour cells demonstrated in the spleen of control and thymectomized Xenopus

Abstract

Summary This paper addresses the issue of natural killer (NK) cell evolution by searching for NK-like activity in an amphibian representative, the immunologically well-characterized clawed frog. Xenopus laevis. Using in vitro 6 h -'chromium release assays, we have shown that splenocyte effectors from early thymectomized (Tx) year-old frogs, but not from control siblings, are able to spontaneously lyse allogeneic thymus tumour cell lines that lack MHC antigen expression. Such lytic capacity can be readily induced in control Xenopus and elevated in Tx frogs by a single injection of tumour cells, with maximal splenocyte cytotoxicity occurring 3 days postinjection, the amount of ^'Cr-release correlating directly with effector: target ratios. Splenocytes, even those from tumour-injected frogs, are unable to lyse allogeneic splenic lymphoblasts or erythrocyte targets, even when the latter are coated with IgY (the .Xenopus IgG equivalent); moreover, we were unable to demonstrate any splenocyte-induced lysis of the human NK cell target K562. Lymphokine-activated killing (LAK) in Xenopus is suggested, since Tx splenocytes cultured in cytokine-rich medium (from concanavalin A-stimulated control splenocytes) display significantly elevated killing of allogeneic tumour targets. Flow cytometric analysis highlights the loss of T cell markers from the spleen of Tx frogs and reveals a variable staining pattern of both control and Tx splenocytes when treated with a mAb that binds to both fish non-specific cytotoxic cells and human NK cells. Prospects for identifying the cellular basis of NK-like activity in Xenopus are discussed in the light of these experiments.

Xenopus NK cells identified by novel monoclonal antibodies

“…Other mAb created against lower vertebrate NK-like cells either fail to label (unpublished data) Xenopus lymphocytes (e. g. the anti-chicken NK mAb 28-4 [27]), or display inappropriate specificity [15] (e. g. the anti-fish NCC mAb 5C6 [8]). …”

“…Spleen, liver and gut lymphocytes were assayed directly ex vivo, or after 2 day preculture. Cells were cultured at 27°C in 5 % CO 2 in 24-well plates, at˚2 × 10 6 leucocytes/ml, in B 3 B 7 tumor cell medium [15] supplemented with 25 % GFM derived from ConA-stimulated X. laevis splenocytes, from which ConA had been removed [17].…”

“…Further analysis of cell surface antigens on sorted NK cells was performed 24 h after separation, and included the use of mAb AM22 (IgM anti-CD8 [43]). Flow cytometry was performed as detailed elsewhere [15,17], gates being set by forward and side scatter to delineate lymphoid cells and exclude dead cells, the latter being confirmed by PI staining. Per sample 5000-10 000 gated cells were analyzed on a Coulter XL flow cytometer.…”

“…Recent studies [15][16][17][18] on control and earlythymectomized (Tx) Xenopus frogs have identified candidate NK cells that display lymphokine-activated natural cytotoxicity towards the MHC-deficient B 3 B 7 lymphoid tumor cell line [19,20]. Since Tx Xenopus frogs are permanently deficient in identifiable T cells and display greatly reduced expression of TCR g transcripts and loss of detectable T cell function [18], they have been used here to generate and screen three mAb that identify and characterize NK-like cells in Xenopus spleen, liver and intestine.…”

Single-cell transcriptome analysis of fish immune cells provides insight into the evolution of vertebrate immune cell types

“…Indeed, mammalian NK-cell receptors have expanded and diversified in a species-specific fashion, as in the case of KIR receptors in primates and Ly49/killer cell lectin-like receptors in rodents (Carrillo-Bustamante et al 2016). NK-like cells have been identified in nonmammalian species such as chicken (Jansen et al 2010), Xenopus (Horton et al 1996), and catfish, where spontaneous killing of allogeneic cells by non-TCR expressing cytotoxic cells was demonstrated (Shen et al 2004;Yoder 2004). A recent study using single-cell qPCR based on known markers of blood cell lineages revealed the presence of a small population of immune cells in zebrafish, which were proposed to represent putative NK-like cells based on expression of NK-lysin genes (Moore et al 2016).…”