Identification of an IL-7-Associated Pre-Pro-B Cell Growth-Stimulating Factor (PPBSF). II. PPBSF Is a Covalently Linked Heterodimer of IL-7 and a M r 30,000 Cofactor

Abstract

Evidence is provided in a companion paper for an IL-7-associated molecular complex that selectively stimulates the proliferation and presumptive differentiation of pre-pro-B cells in our long-term bone marrow culture system and “primes” them to proliferate in response to monomeric IL-7. Here, Western immunoblot analysis reveals that this pre-pro-B cell growth-stimulating factor (PPBSF) is a self-assembling heterodimer of IL-7 and a cofactor with a Mr of 30,000. Thus, when developed with anti-IL-7 mAb, PPBSF migrates electrophoretically as a covalently bound ∼55-kDa molecule under nonreducing conditions but dissociates under reducing conditions. Furthermore, the addition of rIL-7 or native IL-7 to medium conditioned by stromal cells from IL-7 gene-deleted (−/−) mice results in the formation of active 45-kDa and 55-kDa molecular complexes, respectively. Antiserum prepared in IL-7(−/−) mice against affinity-purified PPBSF contained separable reactivities for IL-7 and the non-IL-7 component of PPBSF. The PPBSF cofactor detected by this antiserum migrates as an ∼30-kDa molecule and is able to maintain the viability, but not the proliferation, of pre-pro-B cells. Furthermore, the cofactor is produced constitutively by IL-7(−/−) and IL-7(+/+) bone marrow stromal cells under pro-B- but not pre-B-type culture conditions. Consequently, IL-7 appears to exist almost entirely as a heterodimer (i.e., PPBSF) in pro-B-type cultures, whereas it exists almost entirely as a monomer in pre-B-type cultures. Although the identity of the PPBSF cofactor remains to be determined, it does not appear to be stem cell factor, insulin-like growth factor-1, thymic stromal-derived lymphopoietin, flt3, stromal cell-derived factor-1, or IL-7R.