Development of the Spleen

Lyrnphology 16 (1983) 83- 89 © Georg Thieme Verlag Stuttgart · New York

James F. Jones, M.D.

Department of Pediatrics, University of Arizona College of Medicine. Tucson, Arizona, USA

Summary briefly mention an outgrowth of the meso Embryogenesis and later development of the gastrium invaded by blood vessels and even spleen 's hematologic and immune functions In· tually organized into a loose collection of volves ingrowth of vascular channels, influx of red and white blood cells. Examination of reticular cells and related byproducts to form a filtering network, migration of cells from other individual reports however, reveals a far more organs such as bone marrow and thymus, and sophisticated and highly organized series of final maturation of transient and resident cell events. populations. Barzanji and Emery {1) provide an overview of human splenic embryogenesis. The organ While other animal organ systems subserve fust appears in 8- 10 rnm embryos approxim more than one biological function, the spleen ately 6 weeks into gestation as thickened areas is probably unique in that {I both its hema in the coelomic epithelium of the dorsal me tologic and immune functions rely upon a so sogastrium, which after ingrowth of blood ves phisticated filtering system, (2) parenchymal sels later fuse. Lymphocytes appear by the cells do not "export" activity (i.e. exocrine third month when the organ takes on its char function) and {3) its cellular secretions (e.g. acteristic shape. The spleen grows proportion immunoglobulins arise from cells which have ately with the body to an appropriate size preferentially migrated there. near term. Germinal centers, however, do not form until three weeks after birth even if in Based upon studies of non-human species not utero or perinatal infection supervenes (2). necessarily applicable to man, this treatise propo es that evolution of both hematologic One has to tum to studies in both man and and immune splenic functions depends upon: other species to obtain details of blood vessel , reticular and lymphoid cell development and I. development of mutual vascular channels; organization. At 8.7 weeks blood vessels in 2. influx of reticular cells and their products 38 mm crown-rump length (CRL) human fe to form a filtering network; tuses appear as thin-walled vascular loops; 3. further influx of cells from other organs true arteries veins, and capiiJaries are not re {bone marrow and thymus) to form the cognizable until after the embryo attains a bulk of the mature spleen; and length of 42 rnm. Vasculogenesis is prominent 4. maturation of transient and resident cell in 8.9 - 9.3 week spleens and evolves from populations. a closed loop system into distinct channels opening into sinuses formed by endothelial Embryogenesis cells {3), an arrangement essential to the A general description of embryonic develop spleen's functions. ment of the human spleen is difficult to find. In early spleens, the bulk of the organ is Several well-known embryology texts do not formed from a spongework of branching even index the spleen, while others only connective tissue termed reticular cells. In

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38 mm fetuses, the cells are bulky but tion of reticular and interdigitating cells. For branchless· short branches appear by 42 mm example, experiments in rats show that 5' nu and by 57 nun, slender processes form an cleotidase-bearing reticular cells precede T-cells open meshwork, particularly in the organ's in the periarteriolar areas while acid phosphat center. As these processes progressively enlarge, ase-containing cells arise primarily in germinal the interstitial space among cells widens (3). centers (9). Further organization of white pulp Vessel maturation and reticular cell formation into nodular, transitional and periarteriolar are not independent phenomena, but together sheaths is described but underlying regulating set the stage for development of organized mechanisms are unknown (1 0). areas destined to become white and red pulp. As blood vessels proliferate, reticular cells and their fibers fonn a sheath around developing Hematologic Function arterioles in a pattern similar to that seen in developing lymph nodes (4). During the Oosby (11) enumerates eight red blood cell twelfth through seventeenth week of human (RBC) related functions of the spleen : ery development, a variety of cells invades the thropoiesis, production, cell surface maturation, periarteriolar sheath, including monocytes storage culling, iron metabolism, pitting, and lymphocytes and the dominant cell type, destruction. Periods of development and mech rnacrophages (5). anisms responsible for each function remain elusive, and data derived from both in vivo In the rat these macrophages likely become the interdigitating cells (6) creating the net and in vitro animal experiments may not ap ply to man. For example, erythropoiesis is work for attracting lymphoid cells primarily ofT-lineage in the periarteriolar zone. In fe commonly studied using explants or suspen sions of spleen cells cultured in vitro on chorio tuses with a CRL greater than 98 mm the amniotic membranes (12) or injected into irra marginal zone, formed after macrophage and diated irnmunoincompetent subjects (13). red cell influx, surrounds the periarteriolar zone and continues into the cords of red pulp. Chicken spleen cells by day 15-17 after fertil ization exhibit hematopoietic activity which The red pulp likewise consists of a meshwork disappears at hatching and depends upon cellu of arteries, veins and dark reticular cells into lar aggregates rather than single stem cells. which red cells migrate. Venous sinuses are Repopulation of irradiated mouse spleen or mature by this time, separated from one an bone marrow by spleen cells varies with donor other by cords of marginal zonal tissue. Fe age. In untreated animals spleen cells per gram nestrations in the basement membrane of the body weight is greatest at 12 weeks while sinus suggest that cells move easily from one spleen colony-forming units per gram body compartment to another (see below and Cir weight is maximal at l 0 days and plateaus culatory Dynamics of the Spleen) (5). by 70 weeks. Further differentiation into more specialized Uptake of radiolabelled iron is another tool zones, such as germinal centers, does not take for measuring erythropoiesis. In the rat, ratio place until after birth even though surface im of splenic to bone marrow erythropoiesis is munoglobulin (sig) bearing cells (B cells) and greatest in the weanling and declines slowly erythrocyte rosette forming cells (T -cells) ap thereafter. The reverse is true if the ratio of pear as early as 13 weeks of human gestation body to spleen weight is used suggesting that (7). Differentiation of immune reactive cells erythropoiesis is limited to a fmite period dur does not seem to depend upon antigen stimu ing a stage of slow splenic growth and that lation. Neither children with intrauterine in mature ceU storage or production of other cells fections nor infants born prematurely (8) dis accounts for differences in organism: organ play germinal centers in the spleen until ap weight (14). proximately 3 weeks of age. The process may In man , red cell precursors are found as early depend, however, on formation and matura- as 6-7 weeks of gestation (15). By 8-9 weeks,

Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY. Development of the Spleen 85 maturation is reflected in red cell debris in tion examines immune ontogeny from three newly immigrated macrophages and progeni arbitrary (and perhaps inseparable) viewpoints: tor cells in sinusoids. RBCs in various stages cell identification and description (See also of development as well as mature cells appear Immunoarchitecture of the Human Spleen), in sinusoids by 20 weeks, although the source cell mediated immune function and antibody of these precursor cells is still speculative. The production. presence of progenitor cells among mesenchy T-cell areas in the mouse spleen are purported mal cells removed from the vascular system ly distinguishable from B-cell zones by texture suggests an extravascular origin. Whether and of staining pattern in routinely fixed specimens when true erythropoiesis occurs in man, how (22). In two groups of young adult animals ever, remains unclear. (11 weeks) raised in germ-free environment un Other cells such as granulocytes may also be til 8 weeks, T-cell zones are comparable but produced and undergo maturation in the B-cell areas are slightly larger in one of two spleen. Neonatal mice exhibit high levels of seemingly matched groups, possibly from sea colony-forming cells (180-270/105 ) com sonal differences in the environment. 5 pared with adults (4- 28/10 cells) (16). Surface properties distinguish the various splen Lymphocyte precursors invariably arrive after ic cell populations. Density gradient electropho macrophages (see below) and granulocyte pre resis, a technique based on surface charges of cursors (16, 17). Phagocytic function meas cells, identifies a bimodal distribution of splen ured by technetium sulfacolloid uptake in ro ic lymphoid cells in 3.5 to 5.5 and 7.5 to 17 dents shows the liver to be the chief site of week Balb/c mice but a unimodal distribution uptake for the first 4 weeks of life. Splenic at 6 weeks of age reflecting greater mobility uptake is low at birth and reaches adult levels ofT-cells over B-cells. Thymectomy alters this at ten weeks of age (18). pattern suggesting a thymic influence on sur Studies of red blood cell dynamics and matu face charges (23). ration in developing or neonatal animals are Recently, identification of surface and cyto only rudimentary. Perfusion studies in the plasmic antigens or cell products provides a rat suggest that various populations of red more precise identification of lymphoid cell cells exit the spleen at different times. Fast lineages and maturation patterns in the spleen . washout populations include mature cells Precursor (pre) T- and B-cells are distributed (e.g. peripheral RBCs), while those that wash along with null cells in an age-dependent man out slowly include young cells (58 % reticulo ner in the mouse spleen. Neonatal and 24-week cytes) (19). Reticulocytes preferentially ad mice display the greatest percentage of null here to connective tissues, reticular or inter cells while each of the three groups of cells is digitating cells and sinus walls after release equal in 3 and 8 weeks old mice (24). Other from the bone marrow· and eventually undergo workers trace the origin of pre B-cells to the continued differentiation within the spleen fetal liver and hypothesize migration from (20). This maturation process involves altera that organ to the spleen, where development tion of lipid constituents in the cell membrane continues (25, 26). and is impaired by splenectomy (21). Lymphocytes are noted in the human fetal Immune function spleen at 13 to 15 weeks of gestation or about 6 to 7 weeks after their appearance in the Comparison of age-related events is hampered thymus and blood respectively (27). T-cells by routine use of spleen cells as effector cells (E rosette-forming) constitute the majority in vitro rather than in vivo studies. In this of cells in the thymus and the minority in the system, degree of cellular maturation is un spleen from 13 to 26 weeks of gestation. Al known, and interactions among different cell though only 1 to 2 % of these cells are spleen types cannot be evaluated in their natural en cells before 14 weeks, all immunoglobulin vironment. Within these constraints, this sec- classes as measured by surface immunoglobulin

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(sig) are present in fetuses older than 19 phenomena. Antigen processing must precede weeks. B-cells predominate overT-cells in the T- and B-cell effector reactions. Accessory cells, spleen throughout gestation, the greatest dif primarily macrophages, bearing Ia antigens (ne ference occurring at 20 weeks. cessary for cellular communication) appear and The term T-cells is somewhat misleading as by birth, function in the mouse thymus. Al multiple subsets of cells are able to form E though they are evident in the spleen at 2 weeks rosettes yet function differently. Monoclonal of age, they do not function until weaning (ap antibodies directed against antigens identify proximately 3-4 weeks of age) and presumably these subsets and allow further analysis of therefore cannot stimulate proliferation, matu T -cell ontogeny in the mouse. Lyt-1 denotes ration and activation of resident splenic T-cells helper function, while Lyt-2 and -3 denote until that time (30). cytotoxic-suppressor activity and Thy-1 cor Considerable species variation exists. Some tical thymocytes. Use of these reagents, reveals workers find that murine lymphoid cells bind that peripheral T-lymphocytes derived from a variety of antigens even before birth, although immature thymus cells accumulate in the efferent responses are undetectable until the spleen by one day of age but account for on second week of life (31). Fetal sheep splenic ly 1 % of splenic cells (28). By 2 weeks, how cells also bind antigens as early as 58 days ge ever, the adult frequency of Lyt-1, Thy-1 and station but make no antibodies until consider Lyt-2 bearing cells is already attained. Lyt-1 ably later (32). Antigen binding cells are pres cell number is only slightly greater than Thy-1 , ent in chick embryo spleen on days 14 and 20, while Lyt-2 cells are the least frequent. Thus, associated with an increase in the number of re despite continued growth of the spleen, the ceptors to some but not all antigens. The role total population of T -cell subsets is reached of these unknown antigen binding cells in the by 2 weeks of age and remains constant. embryo remains obscure (33). Other surface antigens serving as important Lymphocyte response to mitogen stimulation markers of lymphocytes are the histocompat is a commonly used though poorly delineated ibility antigens incorporated on the mouse H-2 measure of maturation. In one study, phyto and Ia loci. Newly formed bone marrow lym hemagglutin (PHA) and lipopolysaccharide pro phoid cells in adult mice bear H-2 antigens, voke their greatest response in 4 month old but develop Ia more slowly, the more imma compared to 11 , 20 and 30 month old animals ture cells expressing low density of the anti (34), as the number of lymphoid cells decreases gen. In young postnatal mice, cells bearing la although spleen weight increases. are rare in both bone marrow and spleen, and Similarly, in young mice (3-4 months old) antigen incidence and density do not attain compared to aging ones (26- 30 months), more adult levels in the spleen until 4 and 10 weeks spleen cells but not thymic cells exist in stage respectively (17). G1 of the cell cycle and incorporate 3 H after Enzymes associated with lymphoid cell func concanavalin A (CON A) or PHA stimulation, tion also fluctuate during ontogeny. Adenosine perhaps reflecting the decreasing number of dearninase (ADA) and nucleoside phosphorylase immunoreactive cells in these organs with aging (PNP), enzymes of the purine salvage pathway, (35). are required for normal lymphocyte develop Post-thymic T-cells in neonatal mice are only ment. ADA levels are equivalent in the spleen one-tenth as responsive as adult splenic T-cells of 2, 7 and 24 months old mice; on the other to PHA graft-versus-host production, mixed hand, PNP levels decline steadily from 2 months leukocyte reaction, and in vitro and in vivo B of age and by 24 months are only 63 % of their cell helping, although T-cell cytotoxicity assays earlier peak, possibly related to declining T-cell are comparable to adults. Thymectomy at 5 function with aging (29). days has no influence on expansion and matura As the host matures, changes take place in both tion of post-thymic T-cells (36). afferent and efferent lymphoid cell-mediated

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Lymphocytes mediate yet another function - Production of antibody is one of the most in antibody dependent cell cytotoxicity (ADCC) tensely studied immune functions of the spleen whereby T-cells reorganize antibody on a tar (26, 45), and only the factors influencing de get and proceed to "kill" that target. This velopment of ths phenomenon are addressed function is at a low level in murine spleens here. As previously mentioned, pre-B-cells are at birth, peaks at age 2 months, and then gra present in the spleen, and the number of im dually declines to steady state level during munoglobulin secreting cells varies with age the ensuing 10 months. Removal of phago and species (46) . Some workers propose that cytic cells abrogates the ADCC effect only pre-B-cells must mature within the splenic en in Balb/ c mice (37). virionment before they are able to respond to T-cells themselves are not necessary in vitro non-specific T-cell mediator effects (47). for several functions, and humoral substances Likewise, after T-cells appear in the spleen and such as T-cell replacing factor (TRF) facilitates then develop surface markers, their reactivity is B-cell response to sheep red blood cells (SRBC). curtailed by thymectomy in the immediate While this substance is not present in mouse postnatal period. If neonatal spleen is grafted spleen cell preparations at birth, it emerges by into syngeneic adult animals, cell number and 1- 2 weeks of age (38). suppressor activity are the same as in situ. One explanation for diminished immunocom Spleen graft growth or function in contrast to petence of neonatal compared to adult mouse mitogen and MLC responses is not adversely spleen cells is presence of suppressor cells. affected by host thymectomy. If the recipient Neonatal spleen cells suppress MLC reactions is thymectomized, however, graft spleen devel between adult spleen cells and inhibit antibody opment is enhanced as are cell numbers and production to SRBC (39). In several strains of antibody production (48) . These latter obser mice, an increase in MLC reactivity develops vations suggest that after thymectomy imma as suppressor cell activity diminishes with age. ture helper T-cells selectively increase, and sup In another mouse strain, however, this re pressor cells are "suppressed", suggesting that sponse does not occur suggesting other influ splenic regulatory factors are necessary for nor ences on maturation of this phenomenon (40, mal growth in vivo (49). Additional observations 41). Some evidence suggests that the suppres in aging mice (50) support the contention that sor population does not derive from T -cells, maturation of antibody formation by spleen but rather from miniature macrophages left cells differs from maturation of cell prolifer l:ehind after adherence procedures and that ation. Whether these differences are functional T-lymphocytes are not inhibitors of antibody or due to changing cell populations remains production (42). Nonetheless, in the cow, sup unclear. pression is not even limited to neonates, and a soluble suppressor substance extracted from Conclusion mesenteric lymph nodes of calves greater than The spleen plays an important role in develop four years of age inhibits production of anti ment and maintenance of hematologic and im body by mouse spleen cells in response to thy munologic function in man and animals. Both mus dependent and independent antigens (43). functions depend upon channeling and filtering In vitro differences in B- and T -cell function red and white blood cells as well as foreign between young and old in the mouse are sub particulate material through mutual and/or stantiated in vivo by lack of antibody re separate compartments of the spleen. The vas sponse . as measured by uptake of s1 Cr and cular network established during embryogene 125 1-5-iodo-2-deoxyuridine to intravenous sis and the sequential influx of reticular and SRBC injection in 24 month old in contrast later macrophage derived cells Jay the founda to 3 month old mice. Decreases in both T- tion for subsequent migration of lymphoid and B-cell dependent and independent func and red cells leading to further development tion or an increase in suppressor activity may and maturation of the spleen. explain these results (44).

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While the regulating mechanisms responsible 11 Crosby, W.H.: Normal functions of the spleen for this sequence of events remain sketchy. relative to red blood cells: a review. Blood 14 molecular biology of cell-cell adhesiveness in (1959) 399 12 KeUer, G., C. Havellt. M. Longenecker, E. Diener: embryonic nervous tissue as elucidated by Ontogeny of hemopoietic colony-forming units in Edelman et al. (51) may well apply to the the chick embryo spleen. Adv. Exp. Med. Bioi. spleen, not only providing an explanation 88 (1977) 13 28 for the initiation of events during embryonic 13 Bogg, C., C. Prouskakis: Tire llaemopoieric activi ty of the mouse pleen, at different ages, and its life but also for the continued influx of new response to external and internal irradiation. Br. cells to specific sites throughout the function J. Radio!. 45 (1972) 801 al lifetime of this organ. 14 Aggio, M., N. Guisto, M.l. Bruzzo, M. Montano: The participation of p1een and bone marrow in The nature of mutual interactions among the mice erythropoiesi as a function of age. Acta spleen, bone marrow and thymus is another Physiol. LatlnoAmer. 22 (1972) 1- 5 fruitful area for exploration. Is thls interac 15 Djaldetri, M.: Hemopo1etic events in human em tion simply a matter of shifting cell popula bryonic spleens at early gestational states. Bi.oL Neonate 36 (1979) 133 144 lions or are there humoral mediators? 16 Metcalf, D., S. Stevens: Influence of age and an Studies of the parallel development of hema tigenic stimulation on granulocyte and macro tologic and immunologic function of the phage progenitor cell in the mouse spleen. eU Tissue Kinet. 5 ( 1972) 433 446 spleen may provide some needed answers. 17 Rahal, M.D. , D.G. Osmond: Maturation of bone marrow lymphocyte I . Kinetics of matuntion and renewal of lymphocytes expressing lA and References H-2k Antigen . Immunology 44 (1981) 463 472 18 Ozsoylu, S .• F. Hosain , P.A . Mclntyre: Funcuonal l Bananji, J., J.L. Emery: Change in the pleen development of phagocytic activity of the pleen. related to birth. J. Anat. 129 (1979) J. Pediatr. 90 (1977) 560 562 2 Bananji, J., S.R. Penny, J.L . Emery: Develop 19 Groom, A .C. , S.H. Song, P. Lim, B. Campfing. ment of germinal centers in the spleen in Infant Physical characteri tic of red ceUs coUected from related to birth and unexpected death. J. Clio. the spleen Can. J. Physiol. Pharmacal. 49 (1971) Pathol. 29 (1976) 675 - 679 1092- 1099 3 Weiss, L.: The development of the primary vas 20 Song, S.H., A.C. Groom: Sequestration and pos cular reticulum in the spleen of human fetuses sible maturation of reticulocytes in the normal (38 to 57 mm crownrump length). Am. J. Anat. spleen. Can. J. Physiol. PharmacoL 50 (1972) 136 (1973) 315- 338 400 06 4 Sainte·Marie, G., Y.M. Sin: Reticular fiber pat 21 Shattil, S., R. Cooper: Maturation of macroreti· tern in the rat pleen. Rev. Can. Bioi. 27 culocyte membrane in vivo. J. Lab. Clio. Med . (1968) 273 275 79 (1972) 215 227 5 Weiss, L., L. T. Chen: The differentiation of 22 Meyer, E.M. , W. Schlake, E. Grundman: Compa· white pulp and red pulp in the spleen of hu rative histologic, histochemical and histomorpho man fetuse (72- 145 mm crown-rump length). metric stud1es or T and B ceU areas in penphernl Am. J. Annt. l41 (1974)393 413 lymphoid organs of normal young adult BALB/c 6 WiersbowsA:y, A., V. Grouls, B. Helpop, G. Kling mice. Path. Res. Pract. 164 (1979) 141 - 156 miller' Electron-microscopic tudy of the devel 23 Platsoucos, C.D .. N. Carsimpoolas: Density gra opment of the periarteriolar zone in plenic dient electrophore i of mouse spleen lympho· white pulp of rats. CeU Tissue Res. 223 (J 982) cytes: age-related differences. A critica I Thymu - 335 - 348 dependent event during development in the young 7 Owen, J.J. T., E.J. Jenkinson: Embryology of adult mouse. J . lmmunol. Meth. 34 (1980) 31 41 the lymphoid system. Prog. Allergy 29 (1980) 24 Twomey, J. T. , N.M. Kourltab: The nuU cell com 1 34 partment of the mou e pleen. Cell lmmunol. 63 8 Bananji, AJ., J.L. Emery: Germinal center in {1981) 106 117 the pleen of neonates and stillbirths. Early Hu 25 Beach, R.S .. M.E. Gershwin, R.K. Makisllimo, L. man Development 1 (1978) 363 369 S. Hurley: Impaired immunologic autogeny in 9 Cotayee, G. , M. Clralet, Y. Kochayan · Place des postnatal zinc deprivation. J. utr. 110 (19 0) Cellule Reticularies a Activites Enzymatigur 805 - 815 Oeterminee dans le Developpement de Ia Pulpe 26 Lawton, A .R., M.D. Cooper: Two new s,tage 10 Blanche Sph~ n egue du Rat. Biologie Compte Ren antigen Independent 8-ceU development in mice dus 168 (1974) 1017-1012 and human in Development of Host Defense ed. 10 Milikin , PD.: The nodular white pulp of the hu· M.D. Cooper, D.H. Dayton, Raven Press, New man pleen. Arch. Path. 87 (1969) 247- 158 York 1977, pp 43 54

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27 Hayward, A.R., G. Ezer: Development of lym of neonatal mice. Celllrnmunol. 36 (1978) 354- phocyte populations in the human fetal thy 359 mus and spleen. Clin. Exp. lmmunol. 17 (1974) 40 Lebman, D.A ., C.E. Calkins: Demonstration of 169- 178 active suppressor cells in spleens of young NZB 28 Haaijman , J.J., H.S. Micklem, J.A. Ledbetter, mice. Cell Immunol. 51 (1980) 419 - 423 J.L. Dang/, L.A. Herzenberg: T-cell autogeny: 41 Argyris, B.F.: Role of suppressor cells in immuno Organ localization of maturing populations as logical maturation. Transplantation 31 (1981) defined by surface antigen markers is similar in 334- 338 neonates and adults. J. Exp. Med. 153 (1981) 42 Piguet, P.F. , C. Irle, P. Vassalli: lmmunosuppressor 605 - 614 cells from newborn mouse spleen are macrophages 29 Scholar, E.M., M. Rashidian, M.L. Heidrick: Ade differentiating in vitro from monoblastic precur nosine deaminase and purine nucleoside phospho sors. Eur. J. Immunol. 11 (1981) 56- 61 rylase activity in spleen cells of aged mice. Mech. 43 Delpie"e, M.A., J. Penijel, M. Terquem , J. Maridat: Age Develop. 12 (1980) 323 - 329 Inhibition of in vitro Immune response by factor(s) 30 Lu, C. Y. , D.!. Beller, E.R. Unanue: During onto prepared from lymphoid cells of old animals. Mech. geny, !a-bearing accessory cells are found early Age & Devel. 14 (1980) 181 - 186 in the thymus but late in the spleen. Proc. Nat!. 44 Ansell, J.D ., C.M. McDougall, H.S. Micklem , C.J. Acad. Sci. USA 77 (1980) 1597- 1601 Inchley: Age-related changes in cell localization 31 Spear, P., A . Wang, A . Rutishauser, G. Edelman: and proliferation in lymph nodes and spleen after Characterization of splenic lymphoid cells in fe antigenic stimulation. Immunology 40 (1980) tal and newborn mice. J. Exp. Med. 138 (1973) 687 - 694 557- 573 45 J.R. Battista, J. W. Streilen, ed. : Immuno-aspects 32 Decher, J.M., E.E. Sercarz: Early simultaneous of the spleen. North-Holland Publishing Co. , Am appearance of antigen binding cells in the fetal sterdam 1976, 471 pp sheep. Nature 252 (1974) 416 - 418 46 Benner, R. , A .M. Rijnbeek, R.R. Bernabe, C. Mar 33 Moriya, 0 ., Y. Ichikawa: Ontogeny of spontane tinez-Alonso, A. Coutinho: Frequencies of back ous antigen-binding cells in developing chick em ground immunoglobulin-secreting cells in mice as bryos. Immunology 37 (1979) 857 - 861 a function of organ, age, and immune status. lm 34 Cheung, H.T. , J. Vovolka, D.S. Terry: Age and munobiol. 158 (1981) 255 - 238 maturation-dependent changes in the immune 47 Ito, T., T. Kino, G. Cudkowicz: B-cell differentia system of Fischer F 344 rats. J. Reticuloendo tion in the splenic environment: Acquired suscept thel. Soc. 30 (1981) 563 - 572 ibility to the help function of irradiated T-cells. 35 Joncourt, F. , F. Bettens, F. Kristenser~, A.L. De J. Immunol. 110 (1973) 596 - 599 Week : Age-related changes in mitogen responsive 48 Papiernik, M., S. Ezine: Postnatal development of ness in different lymphoid organs from outbred T-cells. III. Thymus independency of T-cell-depen NMRI mice. lmmunobiol. 158 (1981) 439- 449 dent antigen response in the neonatal spleen. Im- 36 Pigins, P.F., C. Irle, E. Kollatte, P. Vassah : Post munology 46 (1982) 209 - 214 . thymic T-lymphocyte maturation during ontoge 49 Ouedenaren, A . V. , J.J. Haaijman , J. Benner: Fre nesis. J. Exp. Med. 154 (1981) 581 - 593 quencies of background cytoplasmic lg-containing 37 Becker, M.J., J. Roubinian, J.L. Feldmann, M.A . cells in various lymphoid organs of athymic asplen A . Klajman, N. Tala/: Age related changes in anti ic (laset), athymic, asplenic, and normal BALB/c body-dependent cell-mediated cytotoxicity in mice. Immunology 42 (1981) 437- 444 mouse spleen. Israel J. Med. Sci. 15 (1979) 50 Nariuchi, H., W.H. Adler: Dissociation between l47- 150 proliferation and antibody formation by old mouse 38 Boesing-Schneider, 0 .: Functional maturation of spleen cells in response to LPS stimulation. Cell neonatal spleen cells, Immunology 36 (1979) lmmunol. 45 (1979) 295 - 302 527 - 532 51 Edelman, G.M.: Cell adhesion molecules. Science 39 Argyris, B.F.: Suppressor activity in the spleen 219 (1983) 450- 457

James, F. Jones, M.D., Department of Pediatrics, University of Arizona College of Medicine, Tucson, Arizona 85724

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