Functionally all members of the IL
Functionally, all members of the IL-12 family have been annotated along major inflammatory signaling axes but their roles have often weighed on opposing sides of the immunological balance. For instance, IL-12 and IL-23 are classified as pro-inflammatory cytokines  as opposed to the rather protective or regulatory roles of IL-27 ,  and IL-35 , . In fact, all IL-12 family cytokines and cognate receptors have been implicated one way or another in widespread autoimmune and chronic inflammatory disorders including rheumatoid arthritis, psoriatic arthritis, Crohn’s disease, multiple sclerosis, inflammatory bowel disease, and uveitis , , , , , . Thus, it is no surprise that certain members of the IL-12 family emerged as major therapeutic targets, with IL-23 leading the way , . However, the very same principle that defines the structural and functional diversity of the IL-12 family is also the reason why therapeutic targeting of IL-12 family members remains a challenge. The need to differentiate therapeutic targeting of IL-23 from IL to 12 has been highlighted , , and recent clinical approvals of guselkumab and tildrakizumab, two monoclonal 3CAI against IL-23p19 , , , indeed reflect such efforts.
While coverage of the immunoregulatory functions and disease context of IL-12 family cytokines keeps growing, the field is characterized by debilitating gaps in our understanding of structure-function relationships. To date, the only structural and mechanistic data regarding the interaction of an IL-12 family member with a cognate receptor pertains to the very recent crystal structure of the IL-23:IL-23R complex, which together with ancillary biochemical and biophysical undertakings provided insights into how IL-23 becomes structurally primed by IL-23R to mediate a tripartite complex with IL-12Rβ1 . A likely reason for the paucity of structure-function data across the entire IL-12 family might be the lack of biochemically robust explorations of the assembly of heterodimeric cytokine assemblies to fuel their production as recombinant cytokines for biochemical, biophysical, and structural studies. This is particularly problematic for the youngest members of the IL-12 family such as IL-27, IL-35 and IL-39. Here, we tackle this problem by establishing an orthogonal experimental approach that aimed to provide biochemical evidence for the ability of α- and β-cytokine subunits across the entire building block repertoire of the human IL-12 family to interact as secretable recombinant proteins.
Discussion The discovery of IL-12 was a heroic research effort by the teams of Trinchieri and Gately ,  which succeeded in purifying sub-milligram amounts of the then unknown heterodimeric cytokine IL-12 from large volumes of conditioned media (up to 192 L) harvested from primary cell lines. Since the advent of genomic data, strategies of tagging and overexpressing the subunits have become common and all other heterodimeric cytokines of the IL-12 family were reconstituted utilizing a heterologous expression strategy as part of their discovery. Several different strategies of tagging have been utilized over the years to enrich and/or detect specific combination of IL-12 family subunits. In the present study we have utilized a consistent strategy of tagging the subunits that allowed us to interrogate all possible heterodimeric interactions. Furthermore, we tried to minimize the possible deleterious effect the tags could have on the proteins and experimental outcome by reciprocally tagging both termini. The CRLF1 cytokine receptor complexes are not characterized in great detail. The CRLF1:CNTF and CRLF1:p28 complexes would rely on a combination of CNTFRα, LIFR, IL-6R, IL-27R and GP130 although CRLF1 might only play a transient role in these complexes and not be required to engage the actual receptors . We can only speculate at this point about which receptors might form the signaling complex for the possible p35:CRLF1 heterodimer. We propose that the IL-12Rβ2 would likely be involved.