Tuberculin Purified Protein Derivative-Reactive T Cells in Cord Blood Lymphocytes

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1 NFECTON AND MMUNTY, Sept. 1981, p /81/9651-7$2./ Vol. 33, No. 3 Tuberculin Purified Protein Derivtive-Rective T Cells in Cord Blood Lymphocytes HROE SHRATSUCH* AND ZUO TSUYUGUCH Osk Prefecturl Hbikino Hospitl, Osk 583, Jpn Received 26 Jnury 1981/Accepted 4 June 1981 Lymphocytes obtined from cord blood of newborn bbies who were born of helthy mothers were studied in vitro for their responsiveness to purified protein derivtive (PPD) of tuberculin. Cord blood lymphocytes proliferted in vitro by stimultion with PPD, despite wide vritions in the results. Studies with frctionted lymphocytes reveled tht PPD-responding cells belonged to E-rosetting, nylon wool-nondherent T lymphocytes. Non-E-rosetting B lymphocytes lone did not proliferte t ll fter stimultion with PPD. n ddition, bromodeoxyuridine nd light tretment of in vitro PPD-stimulted lymphocytes eliminted the responsiveness to PPD. These results suggest tht T lymphocytes do exist in cord blood nd respond in vitro to stimultion with PPD. A possible role for PPD-rective T lymphocytes in cell-medited protective immunity in newborns is discussed. Antimicrobil protective immunity is brought bout not only by humorl immunity, but lso by cell-medited immunity. mmunity ginst tuberculosis nd some virl diseses is medited mostly by the ction of cellulr immunity. n humns, protective immunity medited by humorl immunoglobulin G, which is pssively trnsferred into the fetus from the mother through the plcent, is known to be operting during the newborn period. On the other hnd, cell-medited immunity hs not been reported to ply n importnt role in protective immunity during tht period. Severl studies on the immunologicl functions of cord blood lymphocytes (CBL) hve been reported previously (7, 9, 15, 16, 21, 25). Most of them were concemed with regultory ctivities in immune responses. The in vitro prolifertive response to purified protein derivtive (PPD) of tuberculin, which is recognized to reflect delyed-type hypersensitivity in vivo (12, 13), hs been investigted in some experimentl systems in CBL (3, 5, 22) or in peripherl blood lymphocytes (PBL) obtined from tuberculin skin test-positive or -negtive individuls (5, 18). However, no evidence hs been reported so fr bout the existence of cell-medited protective immunity during the newborn period. n this present study we demonstrte tht cord blood of newborn infnts contins T lymphocytes which proliferte specificlly in vitro by stimultion with tuberculin PPD. Our results suggest tht cell-medited protective immunity, s well s humorl immunity, my be operting during the newborn period. MATERALS AND METHODS Lymphocyte preprtion. Cord blood smples; were obtined from umbilicl cords t delivery with heprin-coted syringe. All bbies were born of helthy, tuberculin skin test-positive mothers. Lymphocytes were isolted by the Ficoll-Hypque method, s described previously (24). By the E-rosetting method with sheep erythrocytes, it ws reveled tht pproximtely 46% of the CBL thus prepred were E- rosetting T lymphocytes, in contrst to 74% of dult PBL being E-rosetting T lymphocytes. PBL from helthy volunteers were obtined from heprinized venous blood by similr method, using Ficoll-Hypque. The ge distributions of PBL donors were 4 months to 1 yers (men, 3.8 yers) in tuberculin skin test-negtive individuls nd 22 to 67 yers (men, 38.4 yers) in tuberculin skin test-positive individuls. Skin testing ws performed by intrderml injection of pproprite doses of PPD, nd rections were mesured 48 h fter injection. ndividuls who showed >1-mm-dimeter erythem nd indurtion with.5 ug of PPD were regrded s positive; those who showed no rection with s much s 2.5,g of PPD were regrded s negtive. Blood smples were tken before skin testing. Purifiction of T lymphocytes. For the preprtion of purified T lymphocytes, lymphocytes were first rosetted with sheep erythrocytes (E-rosette-forming cells), followed by isoltion by the Ficoll-Hypque sedimenttion technique s described previously (24). Briefly, E-rosette formtion ws crried out bv incubting lymphocytes with sheep erythrocytes for 15 min t 37C, followed by centrifugtion nd nother 12-min incubtion on ice. The mixture of lymphocytes nd sheep erythrocytes ws gently resuspended nd sedimented over Ficoll-Hypque grdient. The E-rosette-forming cells were collected, nd rosetting nd free sheep erythrocytes were lysed in.83% m- 651 Downloded from on September 3, 218 by guest

2 652 SHRATSUCH AND TSUYUGUCH monium chloride. The E-rosette-forming cell-rich frction, fter lysis of erythrocytes, ws then filtered through nylon wool column. The cell suspension ws pplied to the column, incubted t 37 C for 1 h in 5% CO2 in ir, nd then slowly eluted, wshed twice with RPM 164 culture medium (M.A. Bioproducts, Wlkersville, Md.), nd resuspended in the pproprite medium. More thn 95% of the purified T lymphocytes thus prepred rosetted with sheep erythrocytes. They contined less thn 2% surfce immunoglobulin-positive cells. Functionl depletion of dherent cells in the T-cell frction ws scertined by the evidence tht they did not proliferte t ll fter stimultion with concnvlin A or phytohemgglutinin unless reconstituted with plstic petri dish-dherent cells. The non-e-rosetting frction, which floted on the top of the Ficoll-Hypque grdient, ws used s B-lymphocyte-enriched frction. Adherent cells were obtined by recovering the plstic petri dish (Flcon Plstics, Oxnrd, Clif.; no. 32)-dherent non-e-rosetting cells with rubber policemn. Antigens nd lectins. Tuberculin PPD ws kindly donted by H. Fujii, nstitute for Microbil Diseses, Osk University. Tuberculin-ctive peptide (TAP), n cid-extrcted product of mycobcteri (27), ws gift of T. Twr, Toneym Ntionl Tuberculosis Sntorium. TAP is chrcterized s hving the sme ntigenicity s PPD but s lcking immunogenicity when tested with guine pigs (2, 27, 28). Concnvlin A ws purchsed from Miles Yed Ltd., Rehovot, srel, nd pokeweed mitogen ws obtined from GBCO Lbortories, Grnd slnd, N.Y. n vitro ssy of prolifertive response. Lymphocytes were cultured t density of 16 cells per ml in flt-bottomed tissue culture pltes (Microtest, Flcon no. 342) with n pproprite concentrtion of PPD, TAP, or lectins. The culture medium used ws RPM 164 supplemented with 1% pooled humn AB serum, 1 U of penicillin per ml, nd 1,tg of streptomycin per ml. Cultures were performed for vrious times in humidified 5% CO2 in ir t 37 C. Eighteen hours before culture termintion,.2,uci of [3H]thymidine ws dded to ech well. At the end of culture, the cells were hrvested nd wshed, using semiutomted microhrvester (Lboscience Co. Ltd., Tokyo, Jpn), nd the rdioctivity ws counted in Tri-Crb liquid scintilltion counter (Pckrd nstrument Co., nc., Rockville, Md.). Results with cord blood smples whose bckground counts per minutes without PPD or lectin stimultion were s high s 5, were excluded from the dt. Results were expressed s men counts per minute of triplicte determintions, A counts per minute = counts in culture without stimulnt subtrcted from counts in culture with stimulnt, or s stimultion index s follows: counts per minute in culture with stimulnt/counts in culture without stimulnt. BUdR nd light tretment. To eliminte PPDrective lymphocytes, negtive selection procedure with 5-bromo-2-deoxyuridine (BUdR; P-L Biochemicls, nc., Milwukee, Wis.) nd light tretment ws used. A totl of 2 x 16 CBL in 1 ml of culture medium were plced in polystyrene culture tube (Flcon no. 254, 12 by 75 mm) nd cultured in the simultneous NFECT. MMUN. presence of PPD (1 jig/ml) nd BUdR (5 itg/ml) t 37 C in 5% CO2 in ir. On dy 4 of culture, cells were illuminted for 9 min by fluorescent light to eliminte the PPD-induced, deoxyribonucleic cid-synthesizing cells during the culture period. RESULTS PPD-induced prolifertive response of CBL. Time course studies of the PPD-induced in vitro prolifertive response of CBL nd PBL from tuberculin skin test-positive individuls showed tht the mximum prolifertive response of CBL fter stimultion with PPD (1,Ag/ml) ws observed on dy 5 or 6, which ws prllel with PPD-induced prolifertion of PBL from tuberculin-positive donors, lthough the pek response of CBL ws lower thn tht of PBL. PPD doses s high s 2,ug/ml resulted in mximum prolifertive response of CBL ssyed on dy 6 of culture, wheres PBL from tuberculin rectors showed the highest response with PPD doses of 5 ug/ml. The sme doses of TAP (2,ug/ml) were required for CBL to give the mximum response. TAP ws prepred from tubercle bcilli known to hve the sme ntigenicity s PPD. PBL from tuberculin skin testnegtive donors showed no significnt response regrdless of the concentrtion of PPD or TAP used. PPD- or TAP-induced [3H]thymidine incorportion by CBL or by PBL from tuberculin skin test-positive nd -negtive individuls t 6 dys of culture is shown in Fig. 1. The highest men response ws observed in PBL from tuberculin-positive individuls stimulted by either PPD or TAP. Counts per minute of >3 x 14 were observed in nerly hlf of the PBL from tuberculin-positive donors. Two thirds of the CBL subjects tested responded significntly to PPD or TAP (>14 cpm), in contrst to the rections of most of the PBL from tuberculin nonrectors (<13 cpm). The men bckground counts without stimulnt were 2,545 ± 1,749 in CBL, 1, ,12 in tuberculin-positive PBL, nd 1,5 ± 865 in tuberculin-negtive PBL. Age distributions of PBL donors were given in Mterils nd Methods. These results show tht CBL certinly rected to PPD or TAP, lthough the strength of CBL rectivity ws lower thn tht of PBL from tuberculin-positive donors. Prolifertive response of CBL to mitogenic stimultion. Tble 1 shows the response of CBL to nonspecific mitogen stimultion. PPD high- nd low-responding CBL were compred. Lymphocytes were cultured for 3 dys with optiml concentrtions of mitogens. No significnt differences were observed between the PPD high- nd low-responder CBL (Tble 1). Downloded from on September 3, 218 by guest

3 VOL. 33, '-5 x E. u "-4 4 c2 U C3 S._ E 1 C, *16 *1 8 ~ 8-8 *1 1 positive negtive PBL PBL CBL Tuberculin skin test FG. 1. PPD- or TAP-induced prolifertive response of CBL nd PBL from tuberculin skin testpositive or -negtive donors. Lymphocytes were cultured for 6 dys with optiml doses of PPD () or TAP (), nd PHithymidine incorported during the lst 18 h ws counted nd presented s A counts per minute. The men bckground counts were 2,545 ± 1,749 in CBL, 1,951 ± 1,12 in tuberculin-positive PBL, nd 1,5 ± 865 in negtive PBL. PPD-REACTVE T CELLS N CORD BLOOD 653 PPD-induced prolifertive response by frctionted lymphocytes. n humns, the PPD-induced prolifertive response hs been reported to be due to the rections of tuberculinsensitized T lymphocytes to PPD (12, 13), nd nonspecific B-cell mitogenicity hs not been reported so fr. To determine which cell popultion ws proliferting fter stimultion with PPD in CBL, experiments were performed with frctionted lymphocytes. E-rosette-enriched, nylon wool-nondherent cells (T-lymphocyte frction) nd E-rosette-depleted cells (B-lymphocyte frction) were obtined from CBL by the method described in Mterils nd Methods. A totl of 2 x 15 T lymphocytes were cultured together with vrious numbers ofb lymphocytes in the presence of PPD (5 Alg/ml). Tretment of lymphocytes with mitomycin C (4 glg/ml) bolished their bility to proliferte. Figure 2 shows tht PPD-induced proliferting cells were E-rosetting T lymphocytes nd tht non-e-rosetting cells (B lymphocytes) were required for T lymphocytes to respond to PPD stimultion. Next, 2 x 15 B lymphocytes were cultured with vrious numbers of T lymphocytes. The rectivity incresed s the number of T lymphocytes incresed (Fig. 2b). The T- or B-lymphocyte frction lone did not proliferte t ll fter stimultion with PPD. The lck of responsiveness of isolted B lymphocytes to PPD stimultion could be due to the bsence of helper T cells known to be required for B-cell mitogenesis (11). However, ddition of mitomycin C-treted T lymphocytes did not hve n ugmenting effect on the B-lymphocyte response to PPD (Fig. 2 nd b). To exmine the role of non-e-rosetting cells in the PPD-induced prolifertive response of T lymphocytes, dherent cells were obtined by the method described in Mterils nd Methods. Cord blood T lymphocytes were stimulted with PPD or TAP in the presence of dherent cells (Tble 2). T lymphocytes lone did not respond t ll to stimultion with either PPD or TAP. The response of T lymphocytes ws restored by the ddition of dherent cells (T lymphocyte/ dherent cell rio, 1:1). These results showed tht T lymphocytes were responsible for the PPD-induced prolifertive response observed in CBL nd tht dherent cells were essentil for T lymphocytes to respond to PPD. Negtive selection of PPD-rective lymphocytes in CBL. The negtive selection procedure with BUdR nd light tretment ws used to determine whether the CBL response to PPD stimultion represented ntigenic or nonspecific mitogenic rectivity. Depletion of PPD-rective lymphocytes ws crried out by preculturing CBL for 4 dys with PPD in the presence of BUdR. On dy 4 of culture, cultured lymphocytes were illuminted for 9 min with fluorescent light, wshed, nd resuspended in fresh medium. Smples of cells were gin stimulted in vitro for s long s 6 dys with PPD or 3 dys with concnvlin A, nd the prolifertive response ws ssessed by mesuring the mount of [3H]thymidine rdioctivity incorported during the lst 18 h. The results obtined from the 6-dy culture with PPD re shown in Tble 3, nd the dt re expressed s stimultion index. Lymphocytes precultured with PPD in the presence of BUdR followed by light tretment lost rectivity to second PPD stimultion. Lymphocytes treted with PPD only gve seemingly no response when the result ws expressed s stimultion index (Tble 3). They ctully incorported considerble mounts of [3H]thymidine without ddition of PPD in the second culture, however, probbly becuse they Downloded from on September 3, 218 by guest

4 654 SHRATSUCH AND TSUYUGUCH TABLE 1. Prolifertive response of CBL' Acpm (stimultion index) Donors 3-dy culture 6-dy culture PWM ConA PPD TAP PPD high responders 1 51,655 (19.82) 53,142 (2.26) 43,632 (16.38) 2 38,517 (34.82) 2,355 (18.87) 9,54 (1.66) 3,953 (5.2) 3 27,473 (12.91) 11,674 (6.6) 11,358 (3.98) 14,887 (4.91) 4 36,47 (36.37) 11,378 (12.17) 16,932 (11.59) 12,256 (8.66) 5 21,468 (6.43) 15,64 (4.95) 23,338 (17.5) 11,31 (8.78) Men 35,32 (22.7) 22,43 (12.46) 2,965 (11.93) 1,61 (6.84) PPD low responders 6 21,832 (7.4) 3.3 (1.84) 4,2 (2.84) (.89) 7 25,12 (4.86) 15,214 (3.34) 3,53 (2.44) 382 (1.18) 8 25,895 (7.16) 16,54 (4.82) 2,265 (1.82) 1,496 (1.54) 9 38, (15.52) 5,81 (2.41) 1,78 (1.5) 8 (1.37) Men 27,711 (8.64) 21,274 (7.6) 2,599 (2.15) 669 (1.24) CBL (2 x 16) were cultured in vitro for 3 dys with pokeweed mitogen (PWM) (diluted x4) or concnvlin A (ConA) (1,tg/ml) or for 6 dys with PPD (1,tg/ml) or TAP (2,tg/ml). [3H]thymidine incorportion ws expressed s A counts per minute or stimultion index, which is given in prentheses (see text). 2x 15 T cells + B cells b 2x 15 B cells + T cells x E._ No. ct... c B + T T cells dded ( xlo NO. B cells dded x1-5 ) FG. 2. PPD-induced prolifertive response of frctionted CBL. () A totl of 2 x 15 T lymphocytes were cocultured with vrious numbers of B lymphocytes () or mitomycin C-treted B lymphocytes (B,,) () for 6 dys in the presence of PPD (5,ug/ml). Mitomycin C-treted T lymphocytes (T,,) were lso cultured with B lymphocytes (A). (b) A totl of 2 x 15 B lymphocytes were cultured with vrious numbers of T lymphocytes () or mitomycin C-treted T lymphocytes (A) for 6 dys in the presence of PPD (5 pg/ml). Mitomycin C- treted B lymphocytes were lso cultured with T lymphocytes (). were lredy stimulted in the preculture with PPD, nd they showed secondry immune response in the second culture to the smll mount of PPD which ws trnsferred from the first culture. The rectivity of lymphocytes to concnvlin A stimultion, whether treted with BUdR or not, remined intct, lthough the responsiveness ws slightly decresed by 5 NFECT. MMUN. tretment with BUdR. This result rgues for the existence of specificlly PPD-rective lymphocytes in CBL. DSCUSSON n our study we hve demonstrted tht T lymphocytes do exist in cord blood, which proliferted in vitro fter stimultion with PPD or Downloded from on September 3, 218 by guest

5 VOL. 33, 1981 TABLE 2. PPD- or TAP-induced prolifertive response offrctionted CBL PPD TAP Cell popultion S Acpm S Acpm Unfrctionted , ,318 T lymphocytes lone T lymphocytes + d , ,24 herent cells Adherent cells lone.68 Purified T lymphocytes (2 x 16/ml) were stimulted in vitro with n optiml concentrtion (2,ug/ ml) of PPD or TAP in the presence or bsence of 2 x 15 dherent cells per ml. As controls, unfrctionted or dherent cells (2 x 16/ml) were cultured with PPD or TAP lone. Cells were cultured for 6 dys. Results re expressed s stimultion index (S) or s A counts per minute incorported during the lst 18 h of culture. TABLE 3. Negtive selection of PPD rectivity First culture [3H]Thymidine incorportion (stimultion index) BudR PPD Expt 1 Expt 2 Expt 3 Expt CBL were cultured in vitro in the presence (+) or bsence (-) of BUdR (5 jig/ml) or PPD (1 jig/ml) (first culture) for 4 dys, On dy 4, cultures were illuminted for 9 min with fluorescent light. Cells were restimulted with PPD (1 ug/ml) for 6 dys in vitro (second culture). TAP in the presence of dherent cells. Although the optimum dose of PPD required for CBL to show the mximum response ws higher thn tht required for PBL from tuberculin skin testpositive donors, the culture period required to rech the mximum response ws the sme in CBL nd PBL, 5 to 6 dys. The ntigenic specificity of the rection ws shown by the fct tht CBL responded to TAP s well s to PPD. TAP, peptide prepred from tubercle bcilli, is known to hve the sme ntigenicity s PPD but does not hve mitogenicity. Furthermore, the responsiveness of CBL to PPD in vitro ws specificlly eliminted by preculture with PPD nd BUdR tretment. PPD hs been known s B-cell mitogen in mice (1, 23) nd guine pigs (14) nd lso s polyclonl B-cell ctivtor producing immunoglobulins in mice (1) nd humns (6, 8, 19). No evidence hs been reported so fr bout B-cell mitogens of PPD in humns. Our results with frctionted lymphocytes showed tht the PPDinduced prolifertive response in CBL ws elicited by E-rosetting T lymphocytes nd not by PPD-REACTVE T CELLS N CORD BLOOD 655 non-e-rosetting B lymphocytes. TAP is known to hve the sme ntigenicity s PPD (2, 27). We exmined the mitogenic ctivity of TAP in mice. No prolifertive response ws noted when norml mouse spleen cells were cultured together with TAP, in contrst to PPD, which showed strong mitogenic ctivity with mouse spleen cells (dt not shown). However, both TAP nd PPD stimulted prolifertive response of PBL from tuberculin skin test-positive donors, but not from negtive donors. CBL showed similr responsiveness to TAP nd PPD. Frctionted non-e-rosetting cells (B-cell frction) lone did not respond to either PPD or TAP. These observtions fvor the proposl tht CBL proliferted by ntigenic PPD stimultion nd not by PPD nonspecific mitogenic stimultion. The mitogenic ctivity of PPD in CBL prolifertion is lso excluded by the finding tht CBL, whether responding to PPD well or poorly, responded eqully to nonspecific stimultion with concnvlin A or pokeweed mitogen. We lso observed tht PPD-nonresponder PBL proliferted well fter concnvlin A stimultion. One plusible explntion of the PPD-induced prolifertive response of CBL in vitro is tht lymphocytes were primed in vitro nd proliferted fter stimultion with PPD, s hs been reported by Ellner et l. (5). Experiments on in vitro sensitiztion of humn PBL to soluble protein ntigens hve been reported previously (1, 17, 2). According to these reports, optimum priming conditions required t lest 6 or 7 dys of in vitro culture. Our time course study reveled tht the mximum response occurred on dy 5 to 6 of cultivtion, which ws comprble to the PPD-induced prolifertion curve of PBL obtined from tuberculosis ptients. The dely in pek response of CBL in kinetics, s reported by Ellner et l. (5), ws not observed in our study. Another rgument ginst in vitro priming is tht TAP, which is known to lck immunogenicity (28), ws ble to generte prolifertion of CBL in vitro, wheres PBL from tuberculin skin test-negtive donors did not respond to stimultion with either PPD or TAP. Another possibility which cnnot completely be excluded is tht PPD or TAP is functioning s nonspecific mitogen on cord blood T lymphocytes; no findings hve been reported so fr on PPD s T-cell mitogen in humns nd in other nimls. Protective immunity ginst microbil orgnisms is medited by both humorl nd cellulr immunity. During the newborn period in humns, immunoglobulin G ntibodies which were pssively trnsferred into the fetus from the mother through the plcent plyed n impor- Downloded from on September 3, 218 by guest

6 656 SHRATSUCH AND TSUYUGUCH tnt role in protective immunity. Our observtion tht PPD-rective T lymphocytes did exist in cord blood cells strongly suggests tht cellmedited immune mechnisms re lso operting in the ntimicrobil protection during tht period. Generlly, correltion is found between in vitro lymphocyte trnsformtion nd in vivo delyed-type skin rection to tuberculin PPD (4, 12, 13, 19). n newborn bbies whose cord blood cells were studied, skin tests were not performed, lthough ll of the mothers gve positive delyed-type skin rections with ordinry doses of tuberculin PPD. t remins to be elucidted whether PPD rectivity of CBL ws pssively trnsferred from the mother directly, lthough no evidence of pssive trnsfer of lymphocytes from the mother to the fetus vi the plcent hs been reported. A soluble fctor might be trnsferred from the tuberculin-positive mother into the fetus, providing the fetl lymphocytes with rectivity to tuberculin. Studies with CBL from bbies born to tuberculinnegtive mothers will id in elucidting this question, lthough Jpnese re usully vccinted with BCG nd therefore re tuberculin positive. Our preliminry study showed tht no correltion exists between the rectivity of PPDinduced prolifertion of PBL from mothers nd tht of CBL from their bbies. PBL in infnts which showed distinct prolifertive responses to PPD t birth might lose their PPD rectivity soon fter birth. As stted in Mterils nd Methods, children s young s 3 months of ge, unless vccinted with BCG, were PPD nonresponders. ACKNOWLEDGMENTS We thnk Yuichi Ymmur, Osk University, for vluble dvice throughout the study nd Kzuo Ymmoto, Osk Prefecturl Hbikins Hospitl, for criticl review of this mnuscript. We lso thnk Ysuhiro Kwi nd the stff of the Deprtment of Obstetrics nd Gynecology, who provided us with the cord blood smples exmined in this study. LTERATURE CTED 1. Andersson, J., W. Lernhrdt, nd F. Melchers The purified protein derivtive of tuberculin, B-cell mitogen tht distinguishes in its ction resting, smll B cells from ctivted B-cell blsts. J. Exp. Med. 15: Azum,., H. Kimur, T. Niink, T. Aoki, nd Y. Ymmu Chemicl nd immunologicl studies on mycobcteril polyscchrides.. Purifiction nd properties of polyscchrides from humn tubercle bcilli. J. Bcteriol. 95: Bergholtz, B. O., nd E. Thorsby Mcrophge/ T-lymphocyte interction in the immune response to PPD in humns. Scnd. J. mmunol. 9: Blomgren, H Role of B cells in the expression of the PPD response of humn lymphocytes in vitro. Scnd. J. mmunol. 4: NFECT. MMUN. 5. Ellner, J. J., B. Z. Schcter, nd F. T. Bhe Tuberculin response of lymphocytes from humn skin test nonrectors: evidence for in vitro primry sensitiztion of T lymphocytes. Cell. mmunol. 45: Fuci, A. S., nd K. R. Prtt Activtion of humn B lymphocytes.. Direct plque-forming cell ssy for the mesurement of polyclonl ctivtion nd ntigenic stimultion of humn B lymphocytes. J. Exp. Med. 144: Grnberg, C., T. Hirvonen, nd P. Toivnen Cell-medited lympholysis by humn mternl nd neontl lymphocytes: mother's rectivity ginst neontl cells nd vice vers. J. mmunol. 123: Hmmrstroem, L, A. G. Bird, nd C. L. E. Smith Mitogenic ctivtion of humn lymphocytes: protein A plque ssy evlution of polyclonl B-cell ctivtors. Scnd. J. mmunol. 11: Hywrd, A. R., nd A. R. Lwton nduction of plsm cell differentition of humn fetl lymphocytes: evidence for functionl immturity of T nd B cells. J. mmunol. 119: Hensen, E. J., nd B. G. Elferink Primry sensitiztion nd restimultion of humn lymphocytes with soluble ntigen in vitro. Nture (London) 277: Hirno, T., T. Kuritni, T. Kishimoto, nd Y. Ymmur n vitro immune response of humn peripherl lymphocytes.. The mechnism(s) involved in T cell helper functions in the pokeweed mitogen-induced differentition nd prolifertion of B cells. J. mmunol. 119: Jensen, B., M. Kurpisz, nd B. Rubin Antigen specific lymphocyte ctivity in vitro by peripherl blood leucocytes from Mntoux positive nd negtive humn beings.. Comprison of quntittive nd qulittive difference in the PPD-specific lymphoprolifertive response of lymphocytes from two kinds of donors. Clin. Exp. mmunol. 27: Kskur, S Blstogenesis of lymphocytes induced by PPD-primed x-irrdited utologous lymphocytes. J. mmunol. 13: LApsky, P. E., nd A. S. Rosenthl The induction nd regultion of guine pig B-lymphocyte prolifertion in vitro. J. mmunol. 117: Morett, L Active thymus derived suppressor lymphocytes in humn cord blood. Nture (London) 269: Moriy, N., T. Ngoki, N. Okud, nd N. Tniguchi Suppression of dult B cell differentition in pokeweed mitogen-stimulted cultures by Fc(gG) receptornegtive T cells from cord blood. J. mmunol. 123: Newmn, W., G. L. Stoner, nd B. R. Bloom Primry in vitro sensitiztion of humn T cells. Nture (London) 269: Nilsson, B. S The response of lymphocytes from tuberculin-positive or negtive humns to vrious doses of PPD-tuberculin in vitro. Cell. mmunol. 3: Ringden, O., B. Dgoeoe, T. Kunori, A. Freijd, nd E. Moeller Antibody production nd DNA synthesis of humn lymphocyte subpopultions induced by PPD tuberculin. Clin. Exp. mmunol. 36: Rodey, G. E., L. K. Luehrmn, nd D. W. Thoms n vitro primry immuniztion of humn peripherl blood lymphocytes to KLH: evidence for HLA-D region restriction. J. mmunol. 123: Ruusknen, O., W. B. Pittrd m, K. Miller, G. Pierce, R. U. Sorensen, nd S. H. Polmr Stphylococcus ureus Cown -induced immunoglobulin production in humn cord blood lymphocytes. J. mmunol. 125: Schlesinger, J. J., nd H. D. Covelli Evidence for trnsmission of lymphocyte responses to tuberculin Downloded from on September 3, 218 by guest

7 VOL. 33, 1981 PPD-REACTVE T CELLS N CORD BLOOD 657 by brest-feeding. Lncet ii: Sultzer, B. M., nd B. S. Nilsson PPD tuberculin- B cell mitogen. Nture (London) New Biol. 24: Tsuyuguchi,., H. Shirtsuchi,. Terok, nd T. Hirno ncrese in T cells bering gg Fc receptors in peripherl blood of ptients with tuberculosis by in vitro stimultion with purified protein derivtive. Am. Rev. Respir. Dis. 121: Turunen, Significnce of serum fctors in the stimultion of humn umbilicl cord nd fetl lymphocytes by soluble protein A from Stphylococcus ureus. Eur. J. mmunol. 9: Ymmur, Y., K. Onoue, nd. Azum Chemicl nd immunologicl studies on peptide nd polyscchrides from tubercle bcilli. Ann. N.Y. Acd. Sci. 154: Ymmur, Y., K. Onoue, nd T. Thr Purifiction nd the properties of tuberculin ctive peptides. Z. mmunitetsforsch. Allerg. Klin. mmunol. 137: Ymzki, S., K. Koym, S. Somey, L. Azum, nd Y. Ymmur Studies on the llergenicity of vrious tuberculoprotein derivtives nd the djuvnticity ofwx D frctions ofmycobcterium tuberculosis. Am. Rev. Respir. Dis. 1: Downloded from on September 3, 218 by guest