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AUTORADIOGRAPHIC OBSERVATIONS ON THE LOCATION OF DNA— AND RNA— SYNTHESIZING CELLS IN RAT POPLITEAL LYMPH NODES^ by J. J. MILLER III^ (From tlie Walter and Eliza Hall Institute, tlie Department of Exixrimentd Medicine, the University of Melbi)urne, Melbourne, Victoria). (Accepted for publication 1st November, 1964.) Summary. ''H-thymidine and •''H-cytidine have bct'ii usfd to study tlic histohiHical localization of DNA- and RNA-synthesizin^ cells in ptipHteal lymph nodes from untreated, imiuuiiized and ut'onatally tliyineftoinizt'tl rats. Aetive cell proliffration, as measured by •ill-thyinidine incorijoration, was diuiionstrated in tlu' medulla of imstimnlaled nodes from normal and from nconatally tbymeetoniizcd rats. After an immnnolojfical stinuikis, an early increase in medullary cell proliferation was associated with a similar but less intense reaction in the cortex. A partial dissociation between the sites of cell proliferation and the sites of most aetive HNA synthesis was noted. Cells with the highest apparent RNA-syrithetie rates were found predominantly in the cortex. Reticnlar cells, histiocytes, und pliisma eells were found to retain labelled RNA longer than the otlier cells of the lymph nodes. The ability to achieve coasiderably more intense labelling in popliteal lymph node eells than in the rest of the lymphoid system by iiiiid foot-pad injeetions of ''H-nueleosides was confinned. However, attempts to use liiis finding for a study of lympli node eell migration were eomplieateil i>y a failure to olitain adequate numbers of labelled eells after injection of •'H-t!iymidiiic, am! by the diffieiilty t)f fintliiig a dose of •^Il-cytidine wliieli Ialielled small lymphocytes in the popliteal nodes but not blast eells in other organs. It was possible, however, to demonstrate lympliocyte migration within 30 minutes of selective labelling of one popliteal node. Small numbers of migrant cells were found in other lymph nodes, spleen, thymiis, and ileal subniiieosa. INTRODUCTION. The use of ^H-thymidinc to study lymphopoiesis and plasmacytojioiesis in rats has been reported frtjm this laboratory (Nossal UTid Miikela, 1962; Mitchell, MeDonald and Nossal, 1963; Nossal, Mitchell and McDonald, 1963). These 1 This work was supported in part lu' Grant AI-()3y58 from the National Institutes uf Hi'alth, U.S.A., to Dr. C. J. V. Nossal, and in part by a grant from the National Health and Rc'Seareh Conncil. Canberra, to Sir Maefarlunc Bnrnet. -The author was snpported by rost-d<ietora! Grant .5 F2 GM 18, 2fi2-02 from the National Institute.s of Health, U.S.A. Aust. J. exp. Biol. med. Sci. (1965) 4.3, pp. 107-122. 108 j . J. MILLER III studies used autoradiographs of smears aud isolated tells to follow the kinetics of cell proliferation. Aiitoradiographs of smears have also been used to study RNA metabolism iu lymphoid cells (Mitchell, 1964a, 1964b). Since tiiese studies were reported, we have become increasingly interested in the microenvironnieutal aspects of lyuiph node function (Miller and Nossal, 1964). The obscrvations reported in this paper deal with tbe detailed histological location within rat popliteal lymph ntnles of those eells studied on smejirs in earlier reports. MATERIALS AXD METHODS. Animals. Male Wistar albino rats, randomly bred at ttie Hall Institnte, and fed on "Barastoe Dog Gulws", eabbage and tap water ad tihitum were used, l-'ifty rats of varied ages were studied. The details of the different e\periniental eonditions are shown in Table 1. Immunizations. Stoek suspensions of Salmonella adelakh flagellar antigen, prepared as (leserihcd hy Ada, Nossal. Pye, and Abbot (1964), and of proven good antigenieity, were nsed. All injeetions were given subentaneously into the hind foot-pads. Doses of either 10 ug. or 50 fig. per foot-pad were used as shown in Table I. These doses are well ahove the minimum required for an immune response (Nossal, Ada and Austin, 1964), Tritiatcd micleosidcs. ''H-thymidine and ''H-eytidine were used as DNA and RNA precursors respeetivcly. Tliey were obtained from the Radioehemical Gentre, Amersham. England, and consisted of : •'H-thymidine, batch 21, 9-5 euries/millimole, and -'H-cytidine, hateh 9, 1-9 enries/mfllimole, and batch 10, 2'() ciiries/millimole. The nneleosides were injeeted snbeutaneonsly into both or only the left hind foot-pads in O'O.'i to 0-2.5 ml. of saline. The doses, sites and eomses oC injections are shown in Table 1. Neonatallij tlujmvctomizvd rats. Tliree rats from one litter were tliymectomized when less than 24 hours okl. The>' were iinaestlietizetl by heinti placed on iec; cnbes until all movement eeased. The sternnm was split and retracted, the th>inus identified anti remo\e(l by snetion throngh a glass pipette with a 1 mm. orifice. The sternum was elosed with a single silk sntnre, and the overlying skin with fonr interrupted silk sntnres and eoilodion. Similarly treated rats had a redncetl or aKseiit primary re.sponse to Salmonella adelaide ilagellur antigen dnrhig at least the first six weeks of life, hnt gave normal secondary responses (Miller, unpublished). Three litter mates were subjeeted to the same procedure except that the thyinns was not. in fact, removed. At four weeks of age these animals were injeeted with ''H-thymidine intravenously via the dorsal hind foot vein witli the dose indicated in Table 1. Examination of serial seetions of the mediastinal contents re\eaie(l that one of the three "thymectomized" rats had microscopic residual thymif (issue, and tliis animal was excluded from the stiuK'. Pi-cparatkm af uuturudiograph.s. Hats were killed by a hlow on the head nnder ether anaesthesia at varied times after nueleosidc and antigen injection. The popliteal lymph nodes were removed, fixed in 10 per cent neutral formalin for 24 hours, and then dehydrated, cleared, and paraffin emhedtled by standard tcchni<ines. Sections were eiit at 5-7 microns and prepared for autr)racliograpliy witli Kodak AR 10 stripping film nsing the method of Pelc (iy.5G). In must instanecs aiitoiadiograplis of seetions of aortic nodes, mescnteric nodes, thymus. spleen, and ilenm were also prepared. After e.xposnre at 4° for appropriate periods, (ieterniined empirically for each experiment, the autoradiojiraphic film was de\elope(l, fixed and washed, anti the sections were stained with methyl green-pyronin. Extiminathm of autorddiogruiiha. In most instances aiitoradiographs of sections of l)opliteal nodes were simply observed for location iA labelled eells, type of cell labelled, and AUTORADIOGRAPHIC OBSERVATIONS OF LYMPH NODES 109 t: in J: ao 03 be o E 'J£ iS c -^ CS CO . d o V" -f o" x: « c s- c £ 5 ~~ . t ^ ^ . s^ o ;: — £^,-a rf ^' c nun o — IIIUI IIIUI o u *^ 0 kil ?•n 2% in' ••om « « £-; — X til ose iC -a o -n X o c o o c o o o o o o O O o ^. • ^ c g g^ =^ te 3 a. in •"5 It: S O -*• o o o - c o c c c 0 c O c O oo O o c o o Q. s Q. -• K3 O M 0 c o — S > o c o B O O o Tl Tl Tl o O o ^ B , • ' ^ o O H 3 EH d to c — T: £ d S C C d • I., o c td 0 id c id o -1 £ £ E E£ XI H •3 a •v o U o £ a; c -3 -5 o o -a r^ o o Tl Tl T-l GO If- -r lu o t-- •*.. f -rf (M T^ i~ .* (in tn 01 ri 3 •?- rM £ El-. o •ri £ , rt tj' -f -^ mjec 0 Lot onl OIll C 0 o — — o O I-l nject H 0 nject O^ OJ C nject 0 ' O • ^ lie rat Wght) o in 6^ =i S 0 0 • nject c O 0 n nject 7; o - ^ •ri — PQ i ^ dailj days e dail days dailv 0 dav nject O _O C* 0 nject e o CO E! nject ' -^ ti • ^ a a i2 ne .c &a ° o ao >*.-;; • ^ J: nject • -C ^ £ .- njpct nject fll J: r^ J Int *" [;; Q lie 'ri ^ Vi 0 o urae a B s- .. n h CC OJ no J. J. MILLER III relative degree of labelling. In some cases grain counts over labelled cells were determined, and mean values were calculated after counting 40 to 50 cells nf a given type. Similarly, in some cases, the percentage of labelled cells of a given t>'pe was determined hy counting 100 to 200 cells of that type. Organs other than the poplileal ncidcs were examined to determine the degree of labelling of resident cells, and then were scanned for po.ssible migrating cells from the popliteal nodes as previously de.seril)ed (Miller, 1964a). \VIien ''H-eytidine was injected only in the left foot-pad, the right popliteal node was examined for migrant cells. RESULTS. 1. The Location of •''H-thymidinc lucorporafing Cells in Popliteal Nodes. A. Nodes from untreated rats. All tlie normal unstimulated popliteal lymph node.s had labelled large pyroninophilic cells (blast cells) in berth medulla and cortex .sliortly after injection of •4I-thymidine (Eig. 1). Labelled retieular cells and histiocvte.s were less Fij;. 1, An antoradiograph of a section of a normal imstimulatcd 6-7-week-old rat's popliteal lymph node 2)2 hours after a foot-pad injection of •'H-thymidine. Labelled cells, seen as black spots, are present scattered throngh both the medulla and the cortex. (X 150.) common and were mo.st easily distinguished in the medulla and along tbe peripheral sinus. Endothehal cells of the cortical venules and of the efferent lymphatics were oceasionalh" labelled. Labelled mast cells were rare. Small lympboeytes and the rare mature plasma cells were not labelled. AUTORADIOGRAPHIC OHSERVATIONS OF LYMPH NODES IU Those IKKICS fnjm rats which hud received multiple injections of •'*Hthymidine, and ihu.se from rats whieli were killed after periods of mor(^ than one day after nucleoside injection, had small numbers of labelled small lymphocytes tiirouiiliout the cortex. They also had a relatively greater proportion of labelled retitular cells and medullary histiocxtes. Small numbers of labelled mature plasma cells were present in tlie medulla. B. Nodes from antigeniatlhj-stimnhtcd rats. In the primariU- slimuiated rats, the popliteal nodes showed several sequential clianges in the distribution of cells capable of taking up ^H-thymidine following antigen injectiou. liy the seeoud da\ there was an increased density FiR. 2. Till' distrihntinn of labelled cells oO mimites after the injection of -Ul-thymidine on the second da\' ot a niimar>- respon.se is Mcn in this autoracliograpti of a popliteal node. Increased numbers of labelled'ceUs can be seen iu both cortex and medulla, especially in the medullary cords. (X 150.) of labelled cells in both cortex and medulla, but the density was greater in the medulla than in the cortex (Fig. 2). On the tliird day after stimulation, small germinal centres appeared in the eortex. These grew in size until the si.xth day, J. j . MILLEH HI 112 the last time-point includt-d. Most of the cells of these jierniinal centres were lul)elled, hilt usually more lightly than cells in the surnninding cortex. After the third dii\'. iiceiinnilalions of mature plasma cells were found in the medullary cords. These failed to take up 'H-thxiiiidiiie. and tlie density of DNA-synthesizini; cells appeared lower (Fig. 3). Mitotic figures could he found in the medulla throughout the course of the experiment. Fig. !3. An iiiitoradioj-'riipli (it ;i scdioii (if ;i poplitciil node 30 miiuitt'S nftcr ;in injection (if •Ul-tln'[iiidiiK' (Ml tlif fiflli (iiiy of ;i primary rcspmisf. A siiuill H'Tininul triitrf lias appeared in the tortcx. Tin- iiidi\idu;il ci'lls of the utTiiiiiud tcntrc arc not ii.s liiKlilv labelled a.s Cfils rlsewliiTi-. Laryf miinlicrs of Kil)fllcd cells arc still present in the nu'diilla, but the concentration, cell for cell, is less than that seen un the second day of a response (see Kin. 3). (X 150.) The nodes from those rats given •'H-thymidine on tlie third day after antigen, and killed at duiK inter\als until the sixth da\-. showed continued label. gcneralK- witli lower grain counts, in most of the hiast cells of the node. Lahelled small hmphocytes and mature plasma cells appeared in the cortex and medulla respectively. Some of these cells ujipeared iu clumps, but more commonly were scattered siugK. The small Kuiphocytes surrounding gerininal centres never showed sigm'ficant degrees of lahelling. The cells of the genninal centres them- AUTORADIOGRAPHIG OBSERVATIONS OE LYMPH NODES 113 selves showed widely divergent grain counts as time passed, suggesting heterogeneous rates of division. G. Nodes from neonatally thymectoviized rats. The popliteal nodes of the neonatally thymectomized rats showed those changes previously described by Waksman, Arnason and Jankovic (1962). The nodes had thinner cortices than the sham-operated controls, and the internally oriented, large cortical "nodules" were markedly depleted of small lymphocytes. Primary follicles appeared normal in the popliteal nodes. In other lymphoid organs germinal centres and plasma cells were abundant, but small lymphocytes always appeared decreased in numbers. The location of labelled cells after administration of '^H-thymidine was essentially the same as in unstimulated, normal popliteal nodes. There were scattered labelled blast cells in both cortex and medulla (Eig. 4). These cells were particularly frequent Fig. 4. An autoradiograph of a section of a popliteal node from a four-week-old neonatally thymectomized rat one hour after injection of '^H-thymidine. A thinner than normal cortex is present. Labelled cells are again seen scattered through both cortex and medulla. (XlOO.) in the large "nodules" (Eig. 5). Labelled reticular cells and histiocytes were more apparent in the cortex than in the normal nodes. 2. The Location of 'H-cytidine Incorporating Cells in Popliteal Nodes. A. Nodes from untreated rats. Essentially all cells in popliteal lymph nodes were labelled during the first four hours after an injection of ''H-cytidine (Eig. 6). Label appeared largely 114 J. J. MILLER III Fig. 5. A high power view of an autoradioniapli of an iiiUriuil tiutical "nodule" in a popliteal node from a four-week-old neonatally thymectomized rat one hour after injection of ''H-thymidine. The camera was focused on the plane of the grains in the autoradiographic film, thus the cells are not clearly seen. However, tbe relative lack of small lymphocytes can be noted by the size and loose arrangement of the visible nuclei. There are many labelled cells present, inchuliii" on" '" T tvpiral lymph node venulc. (X800.) Fig. 6. An autoradiograph of the cortex of a normal, unstimulated, five-week-old rat's popliteal lymph node one hour after an injection of ^H-cytidine. Label is present in all cell types. The camera was focused on the plane of grains in the autoradiographic film, so cell details are not obvious. The dense, round groups of grains are over blast cells (a). Moderate degrees of labelling can be seen over the venule endothelial cells (b) and reticular cells (c). The small lymphocytes have the least label of those cells seen. (X500.) AUTORADIOGRAPHIC OBSERVATIONS OF LYMPH NODES 115 limited to nuclei. A progression in degree of labelling was apparent: cortical blast cells > reticular cells, histiocytes, and mast cells > small lymphocytes. The blast cells had highly variable counts. The cortical blast cells tended to have higher individual counts than medullary blast cells. Mature plasma cells had the lowest grain counts of any cell present in the nodes and frequently were unlabelled. B. Nodes from antigenically stimulated rats. When ^H-cytidine was given at intervals after primary or secondary antigenic stimulation, and rats were killed shortly after isotope injection, there was an increase in the number of heavily labelled blast cells in both cortex Fig. 7. An autoradiogiai)li ol u .>ti.ii,,,i ,.i .i piipliUal uoJc 31) luinutcs after injection of ''H-cytidine on the second day of a secondary response. The cortex has a concentrated population of heavily labelled cells in contrast to the relative lack of highly labelled cells in the medulla. (X 150.) and medulla (Fig. 7). The number of cells heavily labelled with ''H-cytidine was greater than the number of cells labelled with -'H-thymidine, and these cells tended to be found in greater density in the cortex. Most medullary cells were labelled, but to lesser degrees. Germinal centres which appeared during 116 J. J. MILLER III the course of immune responses had high concentrations of heavily labelled cells (Fig. 8). Germinal centres found in unstimulated nodes did not have such high concentrations of heavily labelled cells, although some were always present. When ^H-cytidine was given on the first day of a primary response, and rats were killed at daily intervals thereafter, a progressively greater proportion of label was found in the cytoplasm of the popliteal lymph node cells. The most Fig. 8. An autoradiograph of a section of a popliteal node 30 minutes after injection of 3H-cytidine on the seventh day of a primary response. A high density of labelled cells is present in the germinal centre, but individual germinal centre cells vary widely in degree of labelling. The difFerence in the concentration of heavily labelled cells in the diffuse cortex as eompared to the medulla is not as marked as is seen in Fig. 7. (X 150.) obvious labelled cells were the dendritic reticular cells and histiocytes of the open sinuses, Cortical cells, both blast cells and lymphocytes, had lost most of their label, but a patterned network of label persisted in the cortex (probably overlying) undefined reticular cell processes. Mast cells and some of the mature plasma cells also retained label, the latter presumably having "matured" in the period after ^H-cytidine injection. This is consistent with a prior report (Miller, 1964b). AUTORADIOGRAPHIC OBSERVATIONS OF LYMPH NODES 117 Fig. 9. An aiitimuliogniph of ii popliteal node from tlie rat which received three injections of •*H-cytidiiie duriiiji a Sfcondary inuniine respiin.sp. Dense labellinil covrrs a germinal centre. Bla.st cells outside tlir gcTininal crntn- tire seen us rnimd, dense groups of grains. The groups of Krains formiiiji monr irregular iiiitterns suggest t]i;it label is persisting jn dendritic reticular cell processes. (\25().) The j)()pl!teal nodes from the rat which w;is killed eight hours after the last of a series of injoctinns of ''Il-cytidine during a second a r\' response had dense labelling over all uerminal centres and over the mc'chilla. The diffuse cortex showed light labelling of most cells with scattered den.sely labelled cells. Some of these were round blast cells, but others appeared to have irregular, dendritic shapes, suggesting that tliey represented reticular cells (ir histiocytes (Fig. 9). Very lightly labelled (ir uiilahelled mature iilasma eells were scattered sinj4y through the medulla (Fig. 10). 3. ObservaHons of Lymphoid Cell Mis,ration. The selective labelling of p{)j-)Iite;il h'mpli imde colls after foot-pad injections of -'H-thymidine, and the use of this teclinicjuc to follow migration of these cells has been reported previously (Miller, 1964a). In the studies reported here, selective labelling of popliteal node cells was also olitained, and migrant cells were found in all lymphoid organs studied, bnt too few for quantitative study. Studies of popliteal lymph node cell migration nsing ''H-cytidine were expected to be more snccessful because more cells, specifically small lymphocytes, were labelled by this nncleoside. However, these studies were compli- 118 j . J. MILU^R 111 Fig. 10. An iiiitnr;uli()gr;i]ih of tlic iiK'diilhi of the .same niiclc jiichircd in Fiy, 9. Note the iiiilabclk'c! liiatiirc plasma cell siirroundfc! hy larj^c iiuin!)ers of heavily labcUed cells. (X200().) eated by the faet that small lymphocytes exhibited much lower grain counts following -'H-cytidine than did blast cells. Thus it was not found possible to label sinall lymphoeytes in one popliteal Kinpli n(Kle without also labelling ijlust cells in the opposite popliteal lymph node to some degree. This is shown ill Table 2, which presents data from four representative experiments. The percentage of blast cells labelled in the left popliteal node (injected side), and TABLK 2. Di.iirihutiaii of hibel nfter injection of ^ff-ri^li'luip Inlo thr. left liiml fool-pnil of uttttimulatcd rats. Doac of nut'lcosido given. [nterval bptween injection iind death Loft jioplitfal node Blast colls Small Iymphooytes Right popliteal node Blast cells % laliplled Menu ^min eomit. ".;, Inbpllpd Mean grain count % labelled Mt'iiii grain count O-.VC/g 30 min 0-5 ^C/g 60 min 98% 100% 73 3 30 mill 60 mill 18'S 1^7 • ( ! 81-5 1'7 :t4 78 8-6 6L' r.4 54 8-6 5 3 U r. • 8 18 :! 7 9-8 AUTORADIOCRAPHIC OBSERVATIONS OF LYMPH NODES 119 their mean grain eounts, are eompared with the same data for the small lymphocytes in the left popliteal node and for the blast cells in the right popliteal node. The grain counts of the small lymphocytes in the right nodes were not significantly above background counts. The ratio of mean grain counts of blast cells ; small lymphocytes, was quite high on the injected side. The mean grain counts of the small lymphocytes in the left popliteal node were slightly higher than the mean grain counts of the blast cells in the right popliteal node. It was therefore thought that study of the migration of small lymphocytes might be possible. A few highly labelled small lymphoeytes could be fcmnd in all lymphoid organs within 30 minntes of injection of -'H-cytidine into one hind foot-pad, and grain count data indicated that these cells were very probably migrants. However, in no instance could this be demonstrated with complete objectivity. The problem is illnstrated in Fig. 11. Moreover, the number of cells found which appeared to be migrants was never large, making significant observations impossible. Fig. 11. An luitoradiiijjriiph of a .section of a right popliteal node from a normal, nnstiniiilatecl rat which had rfceivt'd an injection of -^H-fytidinc in the left hind foot-pad 30 minutes curlier. A cortical veniile is shown. There is a very low degree of labelling of endothelial eells in the veniile ( a ) . Two hl.ist eells adjacent to the vemde hav(^ denser labelling ( h ) . A small cell just oiit.side the \eniile lia.s denser lahe] yet ( c ) . Is the Kinall cell a migrant from the left popliteal node, or was it lahellrd in .situ a.s the endothelial cells and hiast eell prohahly wore? (X 1000.) As far AS they went, the studies of migration using b(jth •'H-thymidine and •'H-cytidine were consistent with eacii other. The cells believed to be migrants in lymph nodes distant from the injection site, were found in the walls of venules or in the adjacent cortex. In the spleen they were found at the border of the lymphocyte cuffs and marginal zones, and less commonly in the red pnlp. In the thymus they were most commonly found in the cortex, occasionally at the 120 J. J. MILLER III cortico-medullary border. In all instances the greatest number of possible migrant cells was found in the subimicosa of the ileum and in the diffuse lymphocyte mass of Feyer's patches. DISCUSSION. These observations support the prior reports on the persistence of small lymphocytes and plasma cells in rat popliteal nodes (Miller, 1964a), the lack of incorporation of RNA precursors into mature plasma cells {Mitchell, 1964a, 1964b), and the stability of plasma cell RNA once it is labelled (Miller, 1964b). They also confirm the obsei-vations of Craddoek, Nakai, Fukuta and Vanslager (1964) on the presence of active ''H-thymidinc incorporating cells in the medulhiry cords of rats, and the relatively low uptake of -^H-thymidine by germinal centre cells in comparison with the blast cells in the difiFuse cortex. Tliese workers believe that germinal centre eells may have a different pattern of DNA metabolism than other lymphoid blast Cflls. An alternative explanation would be that -^H-thymidine has a slower rate of diffusion into germinal centres than into other parts of the lymph nodes. Horowitz, Bauer, Paranetto, Abrams, Watkins and Popper (1964) have reported the location of ''H-thymidine incorporating cells in immunologically reacting lymph nodes in germ-free and "normal" mice. They found that the first increase in numbers of sueh eells occurred in the diffuse cortex, and later an increase appeared in the medulla. In the rats studied in these experiments, this increase occurred simultaneously in cortex and medulla, and the eoneentration of -'H-thymidine ineorjiorating cells was greater in the medulla in the early stages of the primary- response. In both rats and mice active cell proliferation was established in the medulla well before the appearance of germinal centres, indicating that this strueture is not necessary for plasma cell differentiation in a given organ. It remains possible that germinal centime cells from some other lymphoid organ migrated to the popliteal nodes and served as plasma cell precursors. The rat experiments indicated also that germinal centres do not give rise directly to the surrounding lymphocyte cuffs, confirming the findings of Fliedner, Kesse, Cronkite and Robertson (1964). Nossal and Miikela (1962) and Nossal et al (1963) have presented evidence that plasma cells may arise from cells which are actively dividing prior to antigenic stimulation. In the unstimnlated rats studied in tlie.se e.xperiments, proliferating cells, as indieated by ''H-thymidine incorporation, were always present in the medulla. Such cells eould be the stem eells postulated by Nossal's group. On the other hand, none of the popliteal nodes studied here, even from the youngest rats used, was comjiletely free of medullary plasma cells, and it remains possible that the '"'H-thymidine incorporating cells found in the medulla of these rats were present as a part of a naturally-oceuning immunologica] reaction and did not represent a true "stem cell" population. AUTORADIOGRAPHIC OBSERVATIONS OF LYMPH NODES 121 The l(Kation of the most heavily labelled cells after injection of -'H-cytidine appeared to indicate a partial dissociation between the sites of proliferating cells and the sites of cells most active in RNA synthesis. In contrast to the greater concentration of -'H-thymidine incorporating cells in the medulla, those cells most actively incorjiorating 'II-Lytidine were found predominantly in the cortex. Mitchell (1964a, 1964b) demonstrated the relative absence of new RNA s\'ntlit'sis in the maturer elements of tho plasma cell line. The findings reported here would be consistent witli the argument tliat the basic stem cells of the plasma cell line arise in the cortex, where they are active in RNA synthcsi.s, and then move to the medulla, where this activity decreases as the cells "mature". However, there is no proof in any of these oliservations that such a migration occurs. The finding of the labelling of dendritic cytoplasmic processes of reticular cells and liistiocytes at periods of one or more days after the injection of -'Hcytidine indicates that studies of the RNA of tliese cells might be possible in vico. This would be of interest in light of Fishman's (1961) evidence that RNA transfer between histiocytes and lymphocytes is an important step in the initiation of immunological reactions. Achnowlcdgnicnts. The author wishes to thank Dr. G. J. V. Nossal and Sir Macfarlane Burnet for tlieir help and critici.sm of tliis work and puper, and Miss Judy Thompson for painstaking technical assistance. 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