Ancient Cytokine Interleukin 15-Like (IL-15L) Induces a Type 2 Immune Response

Related interleukin-2, -15, and -15-like (IL-2, -15, and -15L) are ancient cytokines, with all three genes surviving in extant fish and some mammals. The present study is the first to identify IL-15L functions, namely in rainbow trout. In isolated trout splenocytes, and in vivo, purified recombinant IL-15L+IL-15Rα molecules induced expression of IL-4 and IL-13 homologs, which are markers of type 2 immunity. In contrast, trout IL-15 stimulated type 1 immunity markers, thus IL-15 and IL-15L can have opposing functions. Trout IL-15L was more dependent on “in trans” presentation by the receptor chain IL-15Rα than IL-15, and stimulated CD4−CD8−(IgM−) lymphocytes from thymus and spleen. We propose an important role for IL-15L early in the type 2 immunity cytokine cascade. Trout IL-2 and IL-15 exhibited features reminiscent of their mechanistic and functional dichotomy observed in mammals; for example, IL-15 but not IL-2 required a receptor alpha chain (only IL-15Rα in the case of fish) for its stability, and only IL-15 was efficient in stimulating lymphocytes from mucosal tissues. Data suggest that IL-15L and IL-15 may be particularly effective in stimulating innate lymphocyte type 2 cells (ILC2) and natural killer (NK) cells, respectively, but further identification of the cell types is needed. An interesting finding different from in mammals was the efficient stimulation of CD4+CD8+ thymocytes by IL-2. In short, this study presents fundamental information on the evolution of the IL-2/15/15L cytokine family.


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Legend to Supplementary  This Supplementary file shows the sequence fragments containing bovine (2A), human (2B), and trout (2C) IL-2, IL-15, IL-15L, IL-2R and/or IL-15R sequences, or modifications thereof, which were cloned into DNA plasmid expression vectors behind a CMV immediate early promoter or into a baculovirus transfer vector behind a p10 promoter (2D). The texts for the individual constructs explain wat the vectors encode, and how the encoded protein is named in the paper. The pink font sequence fragments indicate the restriction sites used for cloning. The DNA plasmid expression vector used for cloning was either pRc/CMV2 (Invitrogen) or pcDNA TM 3.1/myc-His B (Invitrogen). The baculovirus transfer vector used for cloning was pFBD-P10Uhis-ieGFP (a derivate of pFastBac-Dual [Invitrogen], see main text Materials and methods section). The cloning strategies varied and were somewhat arbitrarily because experiments were done at different time points and constructs were made in different laboratories. Several of the expression constructs with bovine IL-2, IL-15L, IL-15R and IL-2R were already described in detail in references (1) and (2), but for convenience are shown here again.
Because the information on the construction method is not so relevant, the below only briefly mentions whether the construction involved an amplification from cDNA or a purchase of a synthetic gene sequence (Invitrogen) and describes the GenBank accession number of a matching sequence. When relevant, additional information is given.
Green font nucleotide sequence: By choice of primer sequences, or synthetic gene ordering, an ACC motif was added directly upstream of the start codon to assure efficient translation.
Red font amino acid sequence: By choice of primer sequences, or synthetic gene ordering, FLAG tag sequences were added.
Orange font amino acid sequence: By choice of primer sequences, or as part of the commercial vector, Myc tag sequences were added.
Purple font amino acid sequence: As part of the commercial vector, poly-His tag sequences were added.
Other colors are explained in the individual texts.

Supplementary file 2A. Expression vectors for (modified) bovine cytokines and receptor chains.
pRcCMV2-Bos-IL-2-FLAG This expression vector encodes bovine FLAG-tagged IL-2 (named IL-2). Cloned from cDNA. The IL-2 part of the sequence is identical with GenBank accession EU276068.
[for cloning details see (1) The part encoding the mature protein was commercially ordered (Invitrogen) with the codons optimized for expression in insect cells, and fused to sequences encoding authentic bovine IL-15L leader sequence and a C-terminal FLAG-tag by using appropriate primers and overlap PCR. The rationale behind the substitutions (shaded yellow) was that in vitro handling of bovine IL-15L protein revealed instability (not shown), while two of the modified motifs were different in mammals from IL-2/15/15L family consensus, and we thought that adding of the trout IL-15La glycosylation motif might aid stability. Unfortunately, an improved stability, or a function, was not found for this IL-15Lhyb molecule.
This expression vector is based on the RLI construct described by Mortier et al. 2006 (3), and encodes a genetic fusion of a human IL-2 leader, a FLAG-tag, a large part of the human IL-15R ectodomain, a glycine-serine linker (shaded blue), and the above described bovine IL-15Lhyb sequence (with the modified motifs shaded yellow) (named bov.IL-15Lhyb-h-RLI). IL-15R does not interact with the signaling receptor IL-2R·IL-2R, and is thought to fixate IL-15 into the proper conformation for binding IL-2R·IL-2R (4); given the observed cross-species activity (main text Fig. 5), we speculated that RLI-versions of bovine and trout IL-15L would have similar stability and signaling advantages as observed for all-human RLI (3,5,6). The linker plus IL-15Lhyb part were commercially ordered (Invitrogen) with the codons optimized for expression in insect cells, and fused to the N-terminal coding part by using plasmid pcDNA3.1-IL-2-Lead-RLI, appropriate primers and overlap PCR.

pcDNA3.1-IL-2-Lead-RLI-trout-IL-15La
This expression vector is based on the RLI construct described by Mortier et al. 2006 (3), and encodes a genetic fusion of a human IL-2 leader, a FLAG-tag, a large part of the human IL-15R ectodomain, a glycine-serine linker (shaded blue), and the rainbow trout IL-15La sequence (named IL-15La-h-RLI; indicated as "RLI" in main text Fig. 8).
The linker plus IL-15La part were commercially ordered (Invitrogen) with the codons optimized for expression in insect cells, and fused to the N-terminal coding part by using the above described plasmid pcDNA3.1-IL-2-Lead-RLI, appropriate primers and overlap PCR.  (3), and encodes a genetic fusion of a human IL-2 leader, a FLAG-tag, a large part of the trout IL-15R ectodomain, a glycine-serine linker (shaded blue), and the rainbow trout IL-15 sequence (named IL-15-RLI).

GGTACCACCATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGT
The sequence was commercially ordered (Invitrogen). The IL-15R part of the sequence is, except for 1 silent nucleotide exchange, identical with GenBank accession GFIN01030410. The IL-15 part of the sequence is identical with GenBank accession NM_001124395.