003 1-3998/94/3503-0289$03.00/0 PEDIATRIC RESEARCII Vol. 35. No. 3. I904 Copyright 0 1994 International Pediatric Research Foundation. Inc. I'r~~t~cll111 L'. S..I.
Human Fetal Antibody-Dependent Cellular Cytotoxicity to Herpes Simplex Virus-Infected Cells
DANIEL V. LANDERS. JANlS P. SMITH, ClIERYL K. WALKER. TERRY MILAb1. LISA SANCHEZ-PESCADOR. AND STEVE KOI1L Dcparit~ic~tlic~/'Oh.vtc~iric~.s. Gj~t~irolo,qj* (cltld Kcprorllictivi~Sc~ic~tri'i:~ ID. I :L.. J.P.S.. C'.K. 11 :. 7.. .\I./i111(1 Ilc8~~c~rtrt~c~tlr ofI'~~(1i~itrics /l*..S-l'., S. K.], Vt~ivc~r.siij~ (~fC~~/i/i~rt~iu, .S~I~IFr~~t~c~i.sco, .S(itl k'rcittci,sc.o Gcttc,r(i/ IIo.s~~ii~i/, S(ltr Frurlc~i.sc'o.Ci~li/i)rtricr 9.11 10
ABSTRACT. Human fetal antibody-dependent cellular cy- not becn clearly demonstrated to be mediated by human fetal totosicity (ADCC) has not beer1 reported previously. hlost cells. Cells from fetal splccn and peripheral fetal blood may investigations have failed to document any cytolptic activity express cell surface antigens characteristic of mature immuno- among fetal lymphocytes. The purpose of this study was cytcs, but limited functional capacity has been identified (1-5). to investigate ADCC activity in the human fetus and iden- In particular, there is very little demonstrated capacity for cell- tify and characterize the effector cell populations in the mediated cytotoxicity among fetal immunocytes in midtrimester. fetus. Fetal spleen cells were separated into single-cell Most information has been derived from umbilical cord blood suspensions and assayed with "Cr-labeled herpes simples samples after delivery. Cell-mediated Iympholysis (CML) has I-infected Chang liver target cells. Significant ADCC ac- becn described in peripheral blood of human fetuses of 18-2 1 tivity was detected in 19 of 26 (73%) of freshly assayed wk gestation. but no such activity was detected in fetal thymus fetal spleen cell preparations from fetuses of 17-24 wk or spleen cells (2-4). No studies have been published that specif- gestational age. This activity, however, was significantly ically address the issue of ADCC during fetal life. less than concurrently run adult peripheral blood mono- The active transport of maternal IgG across the human pla- nuclear cells. After plastic adherence the fetal spleen centa is presumed to play an important role in fctal immune ADCC activity from nonadherent cells was not significantly protection: ADCC activity could be an important defense in different from whole spleen preparations. Surprisingly, situations in which the fetus is exposed to foreign antigens. such ADCC activity in nonadherent fetal cells dropped signifi- as HSV. that infect cells. ADCC is known to play an important cantly after exposure to lates beads, an effect not seen in role in the immune response to HSV infection in neonates after nonadherent adult lymphocytes. Thus, either fetal mono- delivery (5). The present study was designed to determine the cyte-derived (macrophages) fetal spleen cells do not effi- extent of fetal ADCC activity against HSV-infected cells and the ciently adhere to plastic or a unique nonadherent popula- cfTector cell populations responsible for such activity. tion of lates-sensitive imniunocytes is capable of mediating ADCC activity in the fetus. \Ve suspect the former conclu- MATERIALS AND METliODS sion to be the more plausible; however, fluorescence-acti- vated cell sorter staining of fetal cells was not sufficient to Fetal spl~cwccfls. Whole spleens were obtained from 23 nor- confirm these suspensions by fluorescence-activated cell mal human fetuses after elective pregnancy termination bctween sorter analysis. (Pediatr Res 35: 289-292, 1994) 17 and 24 wk gestational age. Spleens were immediately minced and passed through a fine wire-mesh filter, washed in HBSS and Abbreviations subjected to Ficoll-Hypaque differential centrifugation. The mononuclear cell layer was then rcmoved, washed three times ADCC, antibody-dependent cellular cytotosicity in HBSS, and resuspended in minimum essential medium with FACS, fluorescence-activated cell sorter Earle's salts containing 0.05 U/L penicillin, 50 g/L streptomycin, FShlC, fetal spleen mononuclear cell 2 mM glutamine, and 10% heat-inactivated fetal bovine serum HBSS, Ilanks' balanced salt solution (Hyclone Laboratories, Logan, UT) for use as effector cells. HSV, herpes simples virus Iiunratl pcripllrrul blood /~.t)~phoc:~.tc.s.Venous blood samples PBMC, peripheral blood mononuclear cell were obtained aseptically from adult volunteer donors and col- lected in sodium-citrated Vacutainer tubes (Becton-Dickinson. Rutherford, NJ) and immediately subjected to Ficoll-Hypaque differential centrifugation. The mononuclear cell layer was rc- moved, washed three times in HBSS. and resuspended in mini- ADCC is a mechanism whercby Icukocytc effector cells ex- mum essential medium with Earle's salts containing 0.05 U/L pressing the receptor for Ig destroy antibody-sensitive target cells. penicillin, 50 g/L streptomycin, 2 mM glutamine, and 10% heat- ADCC in general, and specifically against viral infected cells, has inactivated fetal bovine serum for use as effector cell controls. Isolrctiot~of tlot~rrrllrcro~~poplr1utiot1.s. Received July 20, 1993; accepted Octobcr 22. 1993. Mononuclear cell sus- Correspondence: Daniel V. Landcrs. M.D.. Dept. OB-GYN. Rm. 6D23. San pensions tvcre obtained as described above. Cells were adhered Francisco General Hospital. 1001 Potrero Ave.. San Francisco. CA 941 10. in six-well 96.2-mm2 plates (Linbro. ICN Flow, Costa Mesa. CA). Supported in part by NIH IPOI-HD24640, HD13021, and AI32384. D.V.L. Approximately 5 x 10h FSMC or adult PBMC tvere plated in 5 was the recipient of an NIII (NICHD) Physician Scientist Award (KI I-HD00750). 1 an American Gynecological and Obstetrical Society Physician Scientist Award mL HBSS and incubated for to 2 h at 37°C in 95% air. 5% (Kennedy-Danreuther). and is currently a Pediatric AIDS Foundation (PAF)/ C02.After incubation, the nonadherent population was removed American Foundation for AIDS Research (AMFAR) Scholar. and the plate washed three times with f1BSS. The cells were 290 LANDER pelleted and resuspended in minimum essential medium. Cell 3% FCS = 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesul- counts were determined and viability verified by trypan blue fonic acid + 0.1 % sodium azide). Cells were counted and viability exclusion. ADCC assays were performed as described below. confirmed by trypan exclusion. FMSC or PBMC were incubated Addition of unfortn laresparticles. For all assays involving the with the manufacturer's suggested concentration of fluoro- use of uniform latex particles, I. 1-pm diameter (Dow Chemical chrome-conjugated anti-CD,, Leu-M I, anti-CD16,or anti-CD56 Co., Indianapolis, IN), the individual cell suspensions were ob- (NKH-I) MAb (Becton Dickinson, Palo Alto, CA). All incuba- tained and treated as described above. The target cells (lo3cells/ tions were performed for 20 min at 2-8°C in the dark. After well), the effector cells (whole cell, nonadherent cell, and adher- incubation, cells were washed three times with cold staining ent cell suspensions at 5 x lo4 cell/well), and latex particles (I0 media and fixed in 2% formaldehyde. Stained cells were kept pL) were added to the assay plate and incubated for I h at 37°C refrigerated and protected from light until analyzed. in 95% air, 5% C02.Control plates (effectors, targets, and 10 pL FACS atlaljsis. Single- and dual-color immunocytometry was media) were also plated and incubated for I h at 37°C. After the performed on the FACS 440 (Becton Dickinson). Ten thousand I-h incubation, HSV I-positive sera, HSV I-negative sera, or events were collected and analyzed using FACSIDESK software media were added to appropriate wells. Plates were then incu- (version 1.9). bated for 18 h (37"C, 5% C02)and harvested as described. Srurislicul atlulj.sis. Data are expressed as the mean + SD. Virus and targel cells. The HE strain of HSV 1 was originally Differences were determined using t test for unpaired samples isolated in the laboratory of Dr. A. J. Nahimias (Emory Univer- for continuous variables and x' or Fisher's exact test for discrete sity, Atlanta, GA). It was propagated in human Chang liver cells variables. using minimum essential medium with Earle's salts containing Ethical cotuidc~rutions.All specimens were obtained after ap- 0.05 U/L penicillin, 50 g/L streptomycin, 2 mM glutamine, and propriate informed consent and within the guidelines set forth 10% heat-inactivated fetal bovine serum. by the University of California, San Francisco Committee on Sera. Human blood was obtained aseptically from volunteer Human Research (IRB). This research did not fall under the donors. After the blood clotted overnight at 4"C, serum was moratorium of the Department of Health and Human Services removed, centrifuged at 200 x gfor 10 min at room temperature, because no therapeutic transplantation of fetal tissue was and inactivated at 50°C for 30 min. All sera were characterized involved. as HSV antibody-positive or -negative by ELISA, Western blot, neutralization, and ADCC assay with known human standards. The HSV-positive sample was characterized as being HSV I+/ RESULTS HSV2- in the laboratory of Dr. Ann Arvin (Department of FShfC rcrslrs adtilt PB,\fC. ADCC activity in this assay system Pediatrics, Stanford University School of Medicine, Palo Alto, was considered significant if it was more than 10% above baseline CA) with an HSV 2 specific IgG ELISA. activity with control (antibody-negative) serum. Significant ADCC assay. As previously described in detail, ADCC was ADCC activity was detected in 19 of 26 (73%) FSMC prepara- determined with HE strain HSV I-infected "Cr-labeled Chang tions of 17-24 wk gestation. ADCC data comparing the means liver target cells approximately 24 h after infection (6-8). Effector of 19 separate fetal spleen cell preparations with significant cells were added to target cells to a final volume of 0.2 mL in activity and PBMC preparations is shown in Figure 1. The mean 96-well round-bottom microtiter plates (Coming Glass Works. ADCC activity detected in fetal spleen cells was 26.6 + 12.9%. Coming, NY). Fetal spleen cells were used at an optimal effector- This activity was significantly less (p< 0.01) than the 39.0 + to-target cell ratio of 50:l as determined by titration curve. 8.6% activity found in concurrently run human adult PBMC. Optimal antibody dilution was determined to be 100: 1 by anti- The presence of ADCC activity in fetal spleen cells did not body titration curve. Effector cells consisted of human fetal correlate with gestational age. spleen cells or adult peripheral blood cells prepared as described Notladll~retrtferal spleen cclls rcJrsfrs~~llole FSAfC. As shown above. Serum known to be positive or negative for anti-HSV in Figure 2, ADCC activity in nonadherent fetal spleen cells antibody was diluted in media and added to wells to achieve a (20.3 + 7.6%) was not significantly different from whole spleen final dilution of 1:100. Assays were harvested after an 18-h preparations (20.7 + 4.7%, p = 0.08). Activity in the adherent incubation at 37°C in 5% C02-enriched air by aspirating 100 pL population (not shown) could not be assessed directly because of from the top of each well without disturbing the cell pellet. To poor viable cell recovery. each well, 100 pL of NaOH was added and the total remaining NonadlrcrenrJ2rul spleen cclls vcrsfis adlrlt t~onadllcrenrPBAfC volume aspirated into a separate container. Each sample was aJer e1-pos1trr to lar~>.~beads. Latex has been shown to be a counted in a Beckman 4000 gamma counter for 1 min (Beckman specific inhibitor of macrophage ADCC in this system (8). Be- Instruments, Fullerton, CA). "Cr release was calculated accord- cause adherent cells normally represent cells of the monocyte- ing to the following formula: % ADCC activity %"Cr release = -2A x 100 A+B where A = cpm in top 100 pL and B = cpm in the pellet to which NaOH was added. ADCC was calculated as follows:
I[(%"Cr release of target cells + effector cells + immune sera) - (%"Cr release of target cells + effector cells + nonimmune sera)] + (100 - %"Cr release of target cells + effector cells + nonimmune sera)l x 100 Fetal Spleen Cells Adult PBMC In this assay system, more than 10% ADCC is significant (7, 8). n=19 n=12 Immunojluorescence staining of cell surjuces. Fetal spleen cell P % ADCC activity 0 Whole Non-Adherent Whole Whole Spken Adult Adult PBML Spleen Cells p=0.8 Spleen Cells S~leen + Beads PBMC + Beads n=10 n=10 n=10 n=8 p