Seminal CD38 is a pivotal regulator for fetomaternal tolerance Byung-Ju Kima,b, Yun-Min Choia,b, So-Young Raha,b, Dae-Ryoung Parka,b, Seon-Ah Parka, Yun-Jo Chunga, Seung-Moon Parkc, Jong Kwan Parkd, Kyu Yun Jange, and Uh-Hyun Kima,b,f,1 + aNational Creative Research Laboratory for Ca2 Signaling Network and Departments of bBiochemistry, dUrology, and ePathology, Chonbuk National University Medical School, Jeonju, 561-180, Korea; cDivision of Biotechnology, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan, 570-752, Korea; and fInstitute of Cardiovascular Research, Chonbuk National University, Jeonju, 561-180, Korea Edited by John J. Eppig, The Jackson Laboratory, Bar Harbor, ME, and approved December 22, 2014 (received for review July 16, 2014) A successful pregnancy depends on a complex process that estab- a variety of inflammatory diseases (20–23). For example, recombi- lishes fetomaternal tolerance. Seminal plasma is known to induce nant CD38 inhibits LPS-induced inflammatory signals in mouse maternal immune tolerance to paternal alloantigens, but the seminal macrophages and human DCs through an interaction with CD31 factors that regulate maternal immunity have yet to be characterized. (24, 25). Here, we show that a soluble form of CD38 (sCD38) released from In this study, we found that CD38 is truncated and released seminal vesicles to the seminal plasma plays a crucial role in inducing into the seminal plasma from seminal vesicles (SVs) as a soluble + + + tolerogenic dendritic cells and CD4 forkhead box P3 (Foxp3 )reg- form (sCD38) in humans and mice. This finding prompted us to ulatory T cells (Tregs), thereby enhancing maternal immune tolerance examine whether CD38 plays a role in maternal immune toler- and protecting the semiallogeneic fetus from resorption. The abortion −/− ance during pregnancy. We found that sCD38 present in seminal rate in BALB/c females mated with C57BL/6 Cd38 males was high plasma was crucial for the induction of uterine tDC and Tregs, compared with that in females mated with Cd38+/+ males, and this + which are responsible for the development of the fetomaternal was associated with a reduced proportion of Tregs within the CD4 tolerance. T-cell pool. Direct intravaginal injection of sCD38 to CBA/J pregnant mice at preimplantation increased Tregs and pregnancy rates in mice Results under abortive sonic stress from 48 h after mating until euthanasia. CD38 Is Truncated and Released from SVs to the Plasma as a Soluble MEDICAL SCIENCES Thus, sCD38 released from seminal vesicles to the seminal plasma acts Form. ADPRC activity was detected in human seminal fluid in as an immunoregulatory factor to protect semiallogeneic fetuses from two proteins with molecular weights of 45 kDa and 37 kDa as maternal immune responses. identified in in-gel assays (Fig. 1A). Notably, the 37 kDa protein bound to a CD38 immunoaffinity column (Fig. 1B, lanes 1 and seminal plasma | CD38 | regulatory T cells | dendritic cells | fetomaternal 3), whereas the 45 kDa protein did not (Fig. 1B, lanes 2 and 4). tolerance This suggests that the 37 kDa protein was a truncated form of CD38, whereas the 45 kDa protein was full-length CD38, which eventy-five percent of pregnancies that are lost represent is encapsulated within prostasomes (13), making the CD38 (45 Sfailure of implantation and are therefore not clinically rec- kDa) physically inaccessible for binding to the affinity column. ognized as pregnancies (1). Recurrent miscarriage (the sponta- Aminoterminal sequencing of the 37 kDa protein identified its neous loss of three or more consecutive pregnancies) is a – cleavage site as Arg58 close to the TM domain of CD38 (Fig. significant health issue for 1 2% of women, with no identifiable 1C). Western blot analysis with two different antibodies against biological cause and no effective treatment. During early stages CD38 peptides (CD3845–57 and CD38171–292) confirmed that this of pregnancy, complex processes help to create a uterine envi- was the case (Fig. 1D). The soluble form, 37 kDa CD38 (sCD38), ronment that is conducive to a successful pregnancy. These was detected in all 19 seminal plasma samples tested (range, include immunological adaptation to the semiallogeneic fetus. Tolerance to paternal alloantigens is critical for successful re- Significance production in placental mammals (2, 3). Many studies have proposed that regulatory T cells (Tregs) play an essential role in the development of fetomaternal tolerance in mice and humans In natural matings, semen delivers spermatozoa and immuno- (4–7). Seminal plasma contains potent immunoregulatory mol- regulatory fluids to the female reproductive tract. Here, a sol- ecules that contribute to the induction of tolerogenic DCs uble form of CD38 (sCD38) is shown to play an important role (tDCs) and ultimately Treg expansion, which is necessary to in facilitating maternal immune tolerance against the fetus by inducing the development of uterine tolerogenic DCs and establish maternal tolerance against paternal antigens (8–10). + + However, the specific molecules in semen that are responsible forkhead box P3 (Foxp3 ) regulatory T cells. Deficiency of for expansion of Tregs and establishment of maternal tolerance sCD38 in seminal fluid increased the rates of loss of allogeneic remain undefined. fetuses, and this loss was rescued by a direct injection of CD38, a mammalian prototype of ADP ribosyl cyclases recombinant sCD38 into the uterus. Thus, seminal sCD38 acts as a pivotal immune suppressor for establishing maternal (ADPRCs), is a type II transmembrane (TM) glycoprotein immune tolerance against the fetus. sCD38 could potentially expressed in many cell types and seminal fluid (11–16). CD38 be used to prevent failed pregnancies. produces calcium-mobilizing second messengers, cyclic ADP ri- bose, and nicotinic acid adenine dinucleotide phosphate (11, 12). Author contributions: B.-J.K. and U.-H.K. designed research; B.-J.K., Y.-M.C., S.-Y.R., We previously showed that intact CD38 in prostasomes assists D.-R.P., S.-A.P., Y.-J.C., S.-M.P., J.K.P., and K.Y.J. performed research; B.-J.K., K.Y.J., + progesterone-induced sperm Ca2 signaling (13). In addition to and U.-H.K. analyzed data; and B.-J.K. and U.-H.K. wrote the paper. 2+ its enzymatic role for Ca signaling, CD38 may also have a The authors declare no conflict of interest. nonenzymatic role through its interaction with CD31 (17, 18). This article is a PNAS Direct Submission. CD31, a type I TM homophilic or heterophilic receptor, is 1To whom correspondence should be addressed. Email: [email protected]. expressed in endothelial cells and a variety of immune cells (19) This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and is involved in attenuating the inflammatory response in 1073/pnas.1413493112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1413493112 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 Cd38−/− b A 1 2 B 1 2 3 4 with or wild-type C57BL/6 (H-2 ) male mice. Mice were examined at 12.5 d postcoitum (dpc), at which time both implan- tation and resorption would be visible. We found that female mice −/− −/− mated with Cd38 males (Cd38 matings) showed higher rates –45 kDa Cd38+/+ –45 kDa –37 kDa of fetal resorption at 12.5 dpc than females mated with –37 kDa P = 0.015 45-57 171-292 AC40 n.s. C 12 12 P = 0.003 10 10 CD38 ID TM ED 30 8 8 58 20 6 6 sCD38 RFPETV 4 4 10 % Resorption 2 = 9 2 = 13 n= 13 n= n= 9 n= n n= 13 n= n= 9 n= 0 n 0 0 D E site implantation Total -/- -/- -/- Viable implantation site Viable implantation Cd38 Cd38 Cd38 HC HC 45 kDa CD38 42 kDa x 40 x 200 x 400 37 kDa sCD38 B 34 kDa Ib Ib Ib jz Epitope : 45-57 171-292 3 mm jz Fig. 1. Identification of sCD38 in seminal fluid by in-gel ADPRC assay. (A) Visualization of human seminal fluid proteins with ADPRC activity (lanes 1 De and 2 for two healthy volunteers). (B) Coomassie blue staining of proteins Wild-typematings 500 m 100 m 50 m 50 m eluted from a CD38 immunoaffinity column (lane 1) and proteins that passed Ib / jz Ib jz through the column (lane 2). In-gel ADPRC activity assay for the proteins 3 mm Ib matings eluted from the column (lane 3) and passed through the column (lane 4). (C) -/- jz Upper diagram shows the intracellular domain (ID), TM domain, and extra- De Cd38 cellular domain (ED) of CD38, and the lower diagram shows the N-terminal 500 m 100 m 50 m 50 m amino acid (aa) sequence (highlighted in red) of sCD38. The segments (aa – – MHCII CD80 P = 0.015 P = 0.04 45 57 and aa 171 292) denoted by green lines represent epitopes of the D 200 60 antibodies used for immunoblotting. The dotted line indicates the cleavage (MFI) 50 (MFI) + 150 site within CD38. (D) Proteins immunoprecipitated from seminal fluid and + 40 prostasome lysates using anti-CD38 antibodies were blotted with antibodies 100 30 – – /CD11c specific for two different epitopes: amino acids 45 57 and 171 292. HC, + /CD11c 20 50 + heavy chain of Ig. (E) Mouse seminal plasma was collected from B6 females 10 Events MHCII by uterine lavage within 1 h of mating with B6 intact (CON), vasectomised CD80 0 1 2 3 4 0 1 2 3 4 0 0 (VAS), and SVX males immunoprecipitated with anti-CD38, and the immu- 10 10 10 10 10 10 10 10 10 10 -/- -/- Isotype Wild-type matings Cd38 Cd38 noprecipitates were analyzed for CD38 by Western blotting. Mouse sple- Cd38-/- matings nocyte lysate (SP) and recombinant mouse sCD38 (sCD38) were included in the experiment as size references of full length and truncated sCD38.