Cloning of cDNA for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on T and natural killer cells.

Abstract

Previously we have reported the purification and characterization of a novel cytokine from an EBV-transformed B cell line, RPMI 8866. This factor, termed natural killer cell stimulatory factor (NKSF), possessed pleiotropic activities including the induction of IFN-gamma from PBL, enhancement of cytotoxicity by NK cells, and stimulation of the proliferation of PBL. Purified NKSF was found to be a disulfide-linked heterodimeric protein composed of 35-kDa and 40-kDa subunits (p35 and p40). We now report the molecular cloning of cDNA for both subunits of NKSF from RPMI 8866 cellular RNA. The cDNA sequences indicate that both genes are novel, and Southern blot analysis confirmed that both cDNA are of human genomic origin. [35S]Methionine labeling indicated that cos-1 cells transfected with either p35 or p40 cDNA produced unique protein species of appropriate size. Methionine labeling of cos-1 cells cotransfected with p35 plus p40 cDNA yielded a broad band migrating between 70 and 90 kDa on a nonreducing gel. Reduction of this high molecular weight material yielded bands correlating with p35 and p40 gene products. Only culture supernatant from cotransfected cos-1 cells had a high level of NKSF biologic activity. That the high molecular weight material was responsible for this activity was indicated by the observation that biologic activity in the culture supernatant migrated at 70 to 90 kDa in a nonreducing gel. Furthermore, anti-p40 serum was able to block the biologic activities of both recombinant and natural NKSF, which indicates that it is a component of the active protein. In contrast, no activity could be detected in the supernatants of cos-1 cells transfected with p40 or p35 cDNA alone. The spectrum of biologic activity produced by cotransfected cos-1 cells was the same as NKSF purified to homogeneity from the RPMI 8866 cell line. A synergistic augmentation of some of these responses was found by the addition of IL-2 or the co-stimulators PHA or phorbol diester. The synergistic stimulation by NKSF plus IL-2 of T and NK function supports the possibility that these cytokines might prove useful in cancer therapy.

Structures of complete extracellular receptor assemblies mediated by IL-12 and IL-23

“…To date, the interaction interfaces between IL-12 and its two cognate receptors had remained poorly understood, in contrast to IL-23:receptor interactions. The herein presented cryo-EM maps reveal the IL-12:receptor interaction interfaces to ˜3 Å resolution (Extended Data Figure 4a,b) and enable headto-head comparisons between the complexes mediated by IL-12 and IL-23 (Figure 2c,d, Extended Data Figure 6,7,8). Although the mIL-12A:IL-12Rβ2 interaction interface is less extensive than the hIL-23A:IL-23R interface (~700 Å 2 vs ∼900 Å 2 ), both interfaces display remarkable commonalities.…”

“…; https://doi.org/10.1101/2023.03. 13.532366 doi: bioRxiv preprint in hIL-23A) (Figure 2c,d ; Extended Data Figure 6,7,8). Notably, mutation of Tyr185 in mIL-12 and the related Tyr189 in hIL-12 fully abolish binding to IL-12Rβ2 18 .…”

“…The N-terminal hook in IL-12Rβ2D1 is much shorter compared to IL-23R and only contacts the tip of α-helix D and stays clear of the loops hanging above it (Figure 2c,d). Furthermore, while IL-23A exclusively interacts with D1 of IL-23R, Gln142 and Asn145 at the tip of helix αB of IL-12A make additional interactions with Asn190 and Arg191 of D2 of IL-12Rβ2, centered around the D1-D2 hinge (Figure 2c,d ; Extended Data Figure 6,7,8). These additional interactions appear to compensate for the shorter N-terminal hook of IL-12Rβ2, which only contributes ~110 Å 2 of interface area, compared to 400 Å 2 for the corresponding N-terminal hook of IL-23R.…”

“…The two founding members of the family, IL-12 and IL-23, serve as pro-inflammatory and pro-stimulatory cytokines in the development of Th1 and Th17 subsets of helper T-cells, respectively 1,5 . Heterodimeric IL-12 and IL-23 share IL-12B (also, termed p40) as their β-subunit, which interacts with IL-12 receptor β1 (IL-12Rβ1) 6 , and are distinguished by their usage of their α-subunits IL-12A (p35) and IL-23A (p19) [7][8][9] , which interact with IL-12 receptor β2 (IL-12Rβ2) and IL23 receptor (IL-23R), respectively 6,10 (Figure 1a). Although the field has been greatly enriched by diverse functional and structural studies [11][12][13][14][15] , essential insights into the architecture of the complete extracellular receptor complexes mediated by…”

Fusion peptide is superior to co-expressing subunits for arming oncolytic herpes virus with interleukin 12

“…In effect, IFN-γ is induced and secreted by these cells. Intact IL-12, p70, is a heterodimeric glycoprotein consisting of two subunits, a 35 kDa light chain (p35) and a 40 kDa heavy chain (p40) 23 , 24 . The p70–IL-12Rβ1 complex binds to the second p70, and the resulting complex then presents a preformed binding site with high affinity for IL-12Rβ2.…”