THE EFFECTS OF THYMUS AND OTHER LYMPHOID ORGANS ENCLOSED IN MILLIPORE DIFFUSION CHAMBERS ON NEONATALLY THYMECTOMIZED MICE

When neonatally thymectomized CBA mice were implanted at 9 to 12 days of age with Millipore diffusion chambers (pore size, 0.1 µ) containing either syngeneic or allogeneic neonatal thymus, they were subsequently found to have the capacity to reject skin homografts and to form antibodies to sheep erythrocytes. In spite of displaying restored immune reactivity, thymectomized mice bearing thymus-filled diffusion chambers still had a lymphopenia and diminished numbers of small lymphocytes in their spleens, lymph nodes and Peyer's patches. Comparison of the lymphoid organs of these mice with those of the thymectomized control mice did not reveal any appreciable difference in the numbers of primary follicles or small lymphocytes. It is postulated that the thymus humoral factor induced immunological competence in lymphoid cells which had left the thymus prior to neonatal thymectomy. The paucity of circulating and tissue small lymphocytes in thymectomized animals, the immune reactivity of which was restored by thymus tissue in diffusion chambers, argues against the theory that the thymus humoral factor has a lymphocytosis-stimulating effect. There was no restoration of immune reactivity in those neonatally thymectomized mice which had been implanted with diffusion chambers containing neonatal or adult spleens, or adult lymph nodes. Thus, the competence-inducing factor is elaborated by the thymus but not by the spleen or lymph nodes. Allogeneic (C57Bl) neonatal thymus tissue, enclosed within diffusion chambers, had the capacity to restore the immune reactivity of totally thymectomized CBA mice, not only to skin homografts of a totally unrelated strain (Ak), but also to grafts isogeneic with the donor of the allogeneic thymus. Therefore, there is no strain barrier to the action of thymus humoral factor. To explain the apparent lack of full participation of thymus lymphocytes in immune reactions it is postulated that thymus lymphocytes are functionally immature in situ, and that they leave the thymus before attaining immunological competence. In the periphery, they undergo further maturation under the influence of the competence-inducing factor produced by the thymus.     

THYMUS-DEPENDENT AREAS IN THE LYMPHOID ORGANS OF NEONATALLY THYMECTOMIZED MICE

Specific areas of lymphocyte depletion, termed thymus-dependent areas, have been delineated in neonatally thymectomized C3H/Bi and F₁ (C57BL x C3H/Bi) mice. They occur within the lymphoid follicles of the spleen immediately surrounding the central arterioles, and constitute the mid and deep cortical zones of the lymph nodes. These depleted areas appear in healthy thymectomized mice as early as 3 wk after operation but, in mice which survive for more than 6 to 7 wk, the thymus-dependent areas are repopulated by rapidly dividing pyroninophilic cells, the majority of which are immature plasma cells. Syngeneic thymus cells, labeled in vitro with tritiated adenosine localize preferentially in the thymus-dependent areas after intravenous injection. Similarly labeled spleen cells also accumulate in these areas but, in addition, are distributed at the periphery of splenic follicles and in the outer cortical zone of the lymph nodes. Many more spleen than thymus cells enter the lymphoid tissues and the spleen appears to be the primary target. The apparent paradox that syngeneic thymus cells are less efficient than spleen cells in restoring neonatally thymectomized mice to normality is discussed in the light of these results and possible routes by which the migrating cells could enter the lymphoid tissues are considered. The origin of the plasma cells which repopulate the lymphocyte depleted areas is also discussed. It is concluded that the normal thymus produces cells which contribute directly to the migratory or circulatory lymphocyte population but that there also exists another source of supply for the plasma cell series. These two systems may function synergistically so that the thymus may control, directly or indirectly, the balance of cell populations within the body.     

ALLOGENEIC THYMUS GRAFTS AND THE RESTORATION OF IMMUNE FUNCTION IN IRRADIATED THYMECTOMIZED MICE

Irradiated and thymectomized CBA mice are markedly depressed in several immunological parameters (skin homograft rejection, graft-vs.-host activity and hemolytic plaque-forming cells of the spleen, hemolysin and hemagglutinin formation, and peripheral lymphocyte counts). In the present experiments the ability of homografts of neonatal thymus placed beneath the kidney capsule to restore immunological capacity of such animals was studied. Thymus homografts which share the same H-2 locus with the CBA mouse were permanently tolerated and immunological restoration was complete. Skin from the thymus donor was specifically retained, but third party skin with even minor (non-H-2) incompatibility was normally rejected and hemolytic plaque-forming cells of the spleen were restored. Thymus homografts which differ at the H-2 locus were promptly rejected and led to accelerated rejection of skin subsequently grafted from the thymus donor. With such H-2 incompatible thymus grafts, third party skin with minor histo-incompatibility was retained while there was slight to moderate restoration of rejection of skin with major (H-2) incompatibility. Graft-vs.-host activity was restored, but there was no return of plaque-forming spleen cells, hemolysins, hemagglutinins, or peripheral lymphocyte counts. In view of the cross-reactivity at the H-2 locus in CBA mice between thymus and third party skin donors, it was felt that restoration of skin rejection and graft-vs.-host activity could be adequately explained on the basis of immunization by the thymus graft and did not require the postulation of true immune restoration or a thymus hormone.     

EFFECTS OF SHORT-TERM EPITHELIAL RETICULAR CELL AND WHOLE ORGAN THYMUS GRAFTS IN NEONATALLY THYMECTOMIZED MICE

Neonatally thymectomized mice were implanted with thymus grafts composed of epithelial reticular cells for periods of 7 and 14 days. Regardless of whether the grafts were placed immediately after thymectomy, or at 3 wk of age, there was little recovery of the lymphocyte depletion and impaired immunologic responsiveness, characteristically found in a neonatally thymectomized host. The findings were similar in animals studied at 2 months or 2 wk after graft removal. Many of the short-term remnant grafts were populated with lymphocytes and had attained the morphologic appearance of thymus by 14 days. A lesser degree of lymphocyte depletion and impaired responsiveness to SRBC occurred if thymectomy was delayed until 7 days of age, if remnant grafts were removed after 2 months, and if intact neonatal thymus was used for the short-term grafts. Complete normality was found in some of the animals in all of these groups. These observations suggest a direct role for mature thymus lymphocytes in reconstituting the neonatally thymectomized host and indicate that epithelial cell function is to direct the maturation of cells that ultimately behave as thymus lymphocytes.     

ROLE OF THE THYMUS IN IMMUNE REACTIONS IN RATS : III. CHANGES IN THE LYMPHOID ORGANS OF THYMECTOMIZED RATS

In rats thymectomized at birth, there was a profound depletion of small lymphocytes in various lymphatic organs. In the spleen, these cells were completely lacking from the Malpighian bodies and splenic white pulp. Empty reticular structures remained surrounding the white pulp arterioles. In the lymph nodes, large masses and nodules of small lymphocytes (primary lymphoid nodules) were either markedly depleted or absent, as were the zones of these cells normally surrounding germinal centers. In both spleen and nodes, germinal centers appeared normal in size, number, and cellular make-up; and plasma cells were found in normal or even increased number in their customary position. Rats which in spite of thymectomy developed intense Arthus or delayed reactivity showed incomplete depletion of the lymphoid tissue. It is concluded that small lymphocytes of the spleen and lymph nodes may come, in large part, directly from the thymus and are not derived from medium and large lymphocytes of the germinal centers. It is suggested that there may be a second population of small lymphocytes whose function is unrelated to the thymus lymphocytes.     

IMMUNOCOMPETENCE OF SPLEEN CELLS FROM NEONATALLY THYMECTOMIZED MICE CONFERRED IN VITRO BY A SYNGENEIC THYMUS EXTRACT

Impaired immunological competence of spleen cells from neonatally thymectomized C57B1/6 young adult mice was apparent when these cells were tested in an in vitro graft-versus-host assay. Spleen cell inocula prepared from thymectomized mice did not induce enlargement of (C3H/eb x C57BI/6)F₁ newborn spleen explants, whereas the same number of cells from intact donors consistently initiated splenomegaly. Spleen enlargement was observed, however, when the explants were challenged by cells from thymectomized donors in the presence of syngeneic thymus extract, indicating that the spleen cells in suspension attained immunological competence under the influence of a non-cellular component of the thymus. Immunocompetence was also evident when the cells from thymectomized donors were first incubated with thymus extract for 1 hr and subsequently tested for reactivity. Cells from the same thymectomized donor mice exposed in parallel to extracts from syngeneic spleen or mesenteric lymph node at an equivalent protein concentration did not initiate a graft-versus-host response. These experiments demonstrate that immune reactivity in the graft-versus-host response involves activation of lymphoid cells by a humoral factor of the thymus acting directly upon these cells.     

CELL TO CELL INTERACTION IN THE IMMUNE RESPONSE : I. HEMOLYSIN-FORMING CELLS IN NEONATALLY THYMECTOMIZED MICE RECONSTITUTED WITH THYMUS OR THORACIC DUCT LYMPHOCYTES

An injection of viable thymus or thoracic duct lymphocytes was absolutely essential to enable a normal or near-normal 19S liemolysin-forming cell response in the spleens of neonatally thymectomized mice challenged with sheep erythrocytes. Syngeneic thymus lymphocytes were as effective as thoracic duct lymphocytes in this system and allogeneic or semiallogeneic cells could also reconstitute their hosts. No significant elevation of the response was achieved by giving either bone marrow cells, irradiated thymus or thoracic duct cells, thymus extracts or yeast. Spleen cells from reconstituted mice were exposed to anti-H2 sera directed against either the donor of the thymus or thoracic duct cells, or against the neonatally thymectomized host. Only isoantisera directed against the host could significantly reduce the number of hemolysin-forming cells present in the spleen cell suspensions. It is concluded that these antibody-forming cells are derived, not from the inoculated thymus or thoracic duct lymphocytes, but from the host. Thoracic duct cells from donors specifically immunologically tolerant of sheep erythrocytes had a markedly reduced restorative capacity in neonatally thymectomized recipients challenged with sheep erythrocytes. These results have suggested that there are cell types, in thymus or thoracic duct lymph, with capacities to react specifically with antigen and to induce the differentiation, to antibody-forming cells, of hemolysin-forming cell precursors derived from a separate cell line present in the neonatally thymectomized hosts.     

STUDIES ON THYMUS FUNCTION : I. COOPERATIVE EFFECT OF THYMIC FUNCTION AND LYMPHOHEMOPOIETIC CELLS IN RESTORATION OF NEONATALLY THYMECTOMIZED MICE

Immunological restoration of 45-day old, neonatally thymectomized C3Hf mice by treatment with humoral thymic function (thymoma grafts, thymus or thymoma in diffusion chambers) ranges from 0 to 12% and is difficult to acheive. When small numbers (5–20 x 10⁶) of young adult lymphohemopoietic cells, ineffective by themselves, are given in association with humoral thymic function, a cooperative effect is observed and restoration ranges from 30 to 60%. With a particular cell dosage (20 x 10⁶), effectivity for cooperation with thymic function was the following in decreasing order: spleen, lymph nodes, thoracic duct cells, bone marrow, blood leukocytes, thymus, and Peyer's patch cells. Comparable results were obtained using spleen, thymus, and hemopoietic liver from newborn donors in association with thymic function. For similar cell dosages, newborn thymus cells were more effective than adult thymus in their cooperative effect with thymic function. Dispersed thymus cells in association with young adult bone marrow or newborn hemopoietic liver cells showed no synergism for the cooperative effect with thymic function in the present model. Using hemiallogeneic cells (F₁ hybrid into parent) it was possible to show that restoration was mediated by proliferative expansion of the injected cells. This was indicated by specific tolerance to tissues of the other parental strain and by cellular chimerism, especially of lymphoid tissues, as indicated by chromosome markers and absence of significant numbers of immunocompetent cells of host origin. A population of paritally differentiated cells of hemopoietic origin, termed postthymic, sensitive to humoral activity of the thymus and present in the lymphohemopoietic tissues of adult and newborn mice is postulated to explain our results. These cells are postthymic and thymus dependent in the sense that they already received thymic influence, probably through traffic, and are incapable of self-renewal in absence of the thymus. Sensitivity to humoral activity of the thymus is characterized by proliferative expansion and/or a differentiative process eventually leading to larger numbers of competent cells.     

STUDIES ON THE RECOVERY OF THE IMMUNE RESPONSE IN IRRADIATED MICE THYMECTOMIZED IN ADULT LIFE

Experiments performed on CBA mice thymectomized in adult life, exposed to lethal doses of irradiation and given tissue therapy are described. Marrow, foetal liver, or spleen cells from syngeneic donors could protect the mice against the lethal effects of irradiation. Between 30 and 70 days' postirradiation, however, marrow-treated, thymectomized irradiated mice showed evidence of trophic disturbances, such as failure to gain weight, in contrast to sham-operated, irradiated, marrow-treated controls. The immune responses of experimental and control mice were tested up to 150 days' postirradiation by challenging with sheep erythrocytes and allogeneic skin grafts. Sham-operated irradiated controls, whether protected with marrow, foetal liver, or spleen cells, produced normal immune responses when challenged at 28, 60, or 150 days after irradiation. Neither foetal liver cells nor marrow cells, in doses of up to 40 million cells per mouse, enabled thymectomized irradiated mice to recover normal immune functions. Spleen cells, from normal donors but not from neonatally thymectomized donors, restored immunological capacity in such mice. It is concluded that immunologically competent cells are present in the spleen of normal adult donors and can function in the absence of the thymus. Bone marrow, on the other hand, does not contain an adequate population of such cells but has lymphoid precursor cells, the descendants of which can become immunologically competent only in the presence of a functioning thymus mechanism.     

THE PRIMARY IMMUNE RESPONSE IN MICE : II. CELLULAR RESPONSES OF LYMPHOID TISSUE ACCOMPANYING THE ENHANCEMENT OR COMPLETE SUPPRESSION OF ANTIBODY FORMATION BY A BACTERIAL ENDOTOXIN

The effects of a single injection of a bacterial endotoxin on the cellular changes of a primary immune response to a standard dose of sheep red blood cells were studied in the spleens and mesenteric lymph nodes of mice. Daily histological comparisons of these organs in mice, injected with endotoxin, or with antigen, or both, showed that endotoxin given simultaneously with sheep red blood cells, as antigen, significantly enhanced all of the cellular changes that appear in the mesenteric lymph nodes and spleens of mice that form antibody when that antigen is given alone. First, in the white pulp of the spleens and cortical regions of the nodes, there appeared an early and excessive proliferation of the large pyroninophilic cells which seems to be responsible for the earliest formation of antibody, as judged by this work and that of others cited in the body of the paper. Polymorphonuclear cells invaded the spleens of these animals early after simultaneous challenge with antigen and endotoxin, and in far greater numbers than have ever been seen in mice given the same antigen without endotoxin. "Activated" germinal centers formed in the lymphoid tissue either 1 day before the appearance of antibody in the blood stream or on the same day, and they became larger than in the mice given antigen only. On the other hand, these specific and characteristic cellular changes failed to appear in mice prevented from forming any antibody at all by injections of endotoxin given 2 days before the antigenic challenge. These findings are discussed in the light provided by data from recent reports of others as well as in the light of the accompanying paper (1) which demonstrated not only the enhancement of antibody formation following simultaneous injections of antigen and endotoxin, as already known, but a totally unexpected, complete suppression of its formation when endotoxin was given 2 days before antigen.     

PROLIFERATION OF DONOR MARROW AND THYMUS CELLS IN THE MYELOID AND LYMPHOID ORGANS OF IRRADIATED SYNGENEIC HOST MICE

CBA/HT₆T₆ bone marrow cells (1 x 10⁷) or CBA/H bone marrow cells (1 x 10⁷) plus CBA/HT₆T₆ thymus cells (5 x 10⁷) were injected intravenously into lethally (800 R) irradiated CBA/H mice. Chromosome analyses of dividing cells in the host lymphoid and myeloid organs were performed at intervals after irradiation. Donor marrow cells settled and proliferated in the host bone marrow, spleen, and lymph nodes soon after injection, but donor marrow cells did not proliferate in the host thymus until day 10; then host-type cells were quickly replaced by donor-type cells in the thymus by day 20. On the other hand, donor thymus cells settled and proliferated in the host thymus and lymph nodes soon after injection but they gradually disappeared from these organs. On day 20, a few donor-type dividing cells (of thymus origin) were found in the host lymphoid and myeloid organs.     

PASSIVE TRANSFER OF TRANSPLANTATION IMMUNITY : I. TRITIATED LYMPHOID CELLS II. LYMPHOID CELLS IN MILLIPORE CHAMBERS

Passive transfer of tritiated thymidine labeled lymphoid cells sensitized to homologous tissues into non-sensitized isologous hosts resulted in accelerated rejection of homologous skin grafts in the recipients. Despite 33 per cent label of the suspension, only rare labeled sensitized lymphoid cells could be found at the site of rejection. Passive transfer of sensitized lymphoid cells in millipore chambers implanted subcutaneously or intraperitoneally in non-sensitized isologous hosts resulted in accelerated homograft rejection in the recipients. Transfer of transplantation immunity could not be accomplished with serum from sensitized hosts. The rejection of homologous tissues without the physical presence of the sensitized cell at the graft site suggested that a humoral agent produced by the cell was capable of rejecting the homograft.     

STUDIES ON THE ROLE OF THE THYMUS IN IMMUNOBIOLOGY : RECONSTITUTION OF IMMUNOLOGIC CAPACITY IN MICE THYMECTOMIZED AT BIRTH

The immunologic competence of spleen cells of mice, as assessed by their graft versus host capabilities, increases to 35 days of age and beyond. Thymectomy at any point along this continuum of development produces "immunologic arrest;" the peripheral lymphoid tissues of such mice do not show significant increases in activity as the animals mature, nor is there appreciable loss of activity up to 6 months after surgery. Adult spleen cells from mice thymectomized at 1 to 24 hours of age have a greatly reduced ability to induce runt disease. Five million spleen cells from immunologically mature animals will uniformly cause fatal runt disease in neonatal recipients, but this same number of cells from neonatally thymectomized animals produces almost no runt disease. When the dosage of cells from neonatally thymectomized C₅₇Bl mice is increased to 20 million, about half of the A recipients develop runt disease. Thus, the defect is a quantitative one. Spleen cells from neonatally thymectomized mice will induce tolerance of skin grafts from the donor strain. In one recalcitrant strain combination, C₅₇Bl to A, use of spleen cells from neonatally thymectomized donors as the tolerance-inducing inoculum permits survival of the recipients, which usually die with severe runt disease, but does not induce tolerance. Cell free extracts of spleen and thymus tissue, including "promine" of Szent-Gyorgyi et al., did not affect the runting syndrome or the immunologic reactivity of neonatally thymectomized mice. When syngeneic thymic tissue is grafted into neonatally thymectomized mice, or the animals are given viable syngeneic spleen or thymus cells, the majority of the animals escape the early mortality characteristic of this group. Administration of syngeneic spleen cells from adult donors and grafting of syngeneic neonatal thymus provide restoration of homograft immunity and graft versus host reactivity of the peripheral lymphoid tissues in most of the neonatally thymectomized animals. Thymus cells rarely provide significant restoration of these parameters. Allogeneic thymus grafts also restore neonatally thymectomized mice. Such animals are chimeric: the immunologically competent cells of their peripheral lymphoid tissues are chiefly of host origin as determined by the discriminant spleen assay, but in many instances a significant donor component is also demonstrable in this system. These chimeric animals accept skin grafts from both donor and host strains. A degree of reconstitution has also been attained by grafting of allogeneic adult spleen in neonatally thymectomized animals. The discriminant spleen assay indicates that cells of the donor strain predominate in the peripheral lymphoid tissues of such mice.     

THE LYMPHOCYTE IN NATURAL AND INDUCED RESISTANCE TO TRANSPLANTED CANCER : VI. HISTOLOGICAL COMPARISON OF THE LYMPHOID TISSUE OF NATURALLY IMMUNE AND SUSCEPTIBLE MICE.

The lymphoid organs of mice show definite changes after cancer inoculation. In immune mice there is a tendency towards a lymphoid hyperplasia, while in susceptible mice more or less marked depletion of the lymphoid tissue takes place. These changes are evident at the end of the 3rd week after cancer inoculation.     

THE EFFECT OF EPITHELIAL REMNANT AND WHOLE ORGAN GRAFTS OF THYMUS ON THE RECOVERY OF THYMECTOMIZED IRRADIATED MICE

Work has been presented which suggests that thymus epithelial reticular cells are not effective in restoring the microscopic morphology of lymphoid tissues and their immunologic capacities. They function in recruiting precursors of thymus lymphocytes from the host animals to produce an organ which, after it becomes architecturally normal, can reconstitute the defective host. Intact thymus grafts in situ from 10–14 days, but not for shorter periods of time, have been shown to result in a return toward normal of these two parameters. Evidence is offered to show that few dividing cellular components in the lymphoid tissue originate from the thymus remnant grafts, and that a minor cellular component is contributed by the intact grafts. These data support the concept that the structural and functional development of the lymphatic tissue in thymectomized animals is dependent on thymus lymphoid cells and/or their products, and that the epithelial-reticular cells do not have a direct action in peripheral lymphoid reconstitution.     

AUTORADIOGRAPHIC ANALYSIS OF LYMPHOPOIESIS AND LYMPHOCYTE MIGRATION IN MICE BEARING MULTIPLE THYMUS GRAFTS

Lymphopoiesis was studied in 3-month-old normal C57Bl mice and in 3-month-old C57Bl mice carrying from 12 to 48 C57Bl thymus grafts using tritiated thymidine labeling. Thymus graft lymphopoiesis was found to be identical with that of normal thymus tissue and the presence of thymus grafts was found to have no influence on host thymus lymphopoiesis. No evidence was found that the massive amounts of thymus graft tissue in the mice affected any parameter of host lymph node lymphopoiesis nor was any evidence detected for the migration of thymic lymphocytes from these massive deposits of thymus graft tissue either to host lymph nodes and blood or to other organs in the host animal. It is concluded that the majority of small lymphocytes produced in the thymus and thymus graft tissue do not migrate from these tissues but die locally at the end of their intrathymic life span of 3 to 4 days.     

CELLS INVOLVED IN THE IMMUNE RESPONSE : IV. THE RESPONSE OF NORMAL AND IMMUNE RABBIT BONE MARROW AND LYMPHOID TISSUE LYMPHOCYTES TO ANTIGENS IN VITRO

Cell suspensions of immune rabbit lymph nodes and spleen were capable of undergoing blastogenesis and mitosis and of incorporating tritiated thymidine when maintained in culture with the specific antigen in vitro. They did not respond to other, non-cross-reacting antigens. The blastogenic response obtained with immune lymph node cells could be correlated with the antibody synthesizing capacity of fragment cultures prepared from the same lymph nodes. Cell suspensions of immune bone marrow responded to non-cross-reacting antigens only whereas cell suspensions of immune thymus, sacculus rotundus, and appendix did not respond when exposed to any of the antigens tested. On the other hand, neither fragments nor cell suspensions prepared from lymph nodes, spleen, and thymus of normal, unimmunized rabbits responded with antibody formation and blastogenesis when exposed to any of the antigens. However, normal bone marrow cells responded with marked blastogenesis and tritiated thymidine uptake. The specificity of this in vitro bone marrow response was demonstrated by the fact that the injection of a protein antigen in vivo resulted in the loss of reactivity by the marrow cell to that particular antigen but not to the other, non-cross-reacting antigens. Furthermore, bone marrow cells of tolerant rabbits failed to respond to the specific antigen in vitro. It was also demonstrated that normal bone marrow cells incubated with antigen are capable of forming antibody which could be detected by the fluorescent antibody technique. This response of the bone marrow cells has been localized to the lymphocyte-rich fraction of the bone marrow. It is concluded that the bone marrow lymphocyte, by virtue of its capacity to react with blastogenesis and mitosis and with antibody formation upon initial exposure to the antigen, a capacity not possessed by lymphocytes of the other lymphoid organs, has a preeminent role in the sequence of cellular events culminating in antibody formation.     

VIRUS PARTICLES IN THE THYMUS OF CONVENTIONAL AND GERM-FREE MICE

Electron microscope study of thymuses of both conventional and germ-free mice has revealed the presence of typical virus particles associated with the thymic lymphocytes or with the thymic epithelial cells. The particles resemble those associated with several murine leukemias and their viral nature seems convincingly substantiated by morphological observation. Germ-free mice are therefore not virus-free. The biological significance of these particles is still unknown and we can only speculate as to the possible relationship of these particles to the incidence of "spontaneous" leukemia, to the lymphocytosis stimulating factor of Metcalf, and to the numerous latent viral infections of laboratory mice.     

THE ROLE OF THE THYMUS IN DEVELOPMENT OF IMMUNOLOGIC CAPACITY IN RABBITS AND MICE

In rabbits, complete thymectomy before the age of 5 days produced immunologic deficiency in the adult animals, as indicated by reduced antibody production to bovine serum albumin and bacteriophage T₂. Homotransplantation immunity was unaffected, however. In an inbred strain of mice, complete neonatal thymectomy resulted in complete inability of the 60-day-old animals to form antibody to bacteriophage T₂. Inbred mice, completely thymectomized at birth, had a deficient homograft response, indicated by acceptance of skin homografts from strains differing in both the weaker and stronger (H-2) histocompatibility antigens. Tumor transplants (mammary adenocarcinoma) were also successful across the H-2 genetic barrier in mice thymectomized at birth. Neonatal thymectomy also eliminated the Eichwald-Silmser phenomenon, rendering female mice capable of accepting isografts of male skin. Transplantation immunity in mice was also affected by later thymectomy, at 30 days of age, in certain strain combinations involving weak histocompatibility differences. Spleen and lymph node cells from mice thymectomized at birth or at 6 days of age, and sacrificed 2 months later, did not produce a graft versus host reaction in appropriate F₁ hybrid recipients, indicating that such cells are immunologically inactive. Neonatal thymectomy of F₁ hybrid mice, and in one strain combination thymectomy at 40 days of age, produced animals with inordinate susceptibility to runt disease (homologous disease) following injection of parent strain spleen cells 35 days (neonatal surgery) and 10 days (surgery at 40 days) later. Mice thymectomized at birth also showed growth failure and were short-lived. Studies of newborn mice indicated that they have true lymphocytes only in the thymus, and lack such cells in the spleen, lymph nodes, and gut. In normal mice, adult lymphoid structure develops gradually, beginning during the 1st week of life and continuing for the next month. In contrast, mice thymectomized at birth do not develop mature lymphoid structure: the lymph nodes and spleens tend to be small and poorly organized, and show a quantitative deficiency in lymphoid cells. It is our current working hypothesis that the thymus makes a major contribution toward the centrifugal distribution of lymphoid cells which, in turn, is essential to the full expression of immunologic capacity.     

MATURATION OF HEMOLYSIN-PRODUCING CELL CLONES : II. THE APPEARANCE AND LOCALIZATION OF PRECURSOR UNITS IN LYMPHOID TISSUES OF NEONATAL MICE

Cells capable of reacting with sheep erythrocyte (SRBC) antigen to maturate and produce hemolysin appear simultaneously in the bone marrow and spleen of 1-day old Swiss-Webster mice. However, hemolysin-producing cell clones (HPCC) do not result. Complete functional precursor units generally appear in the spleens of mice older than 3 days. In vivo and in vitro data correlate well in this regard. Complete precursor units are not seen in the bone marrow and only very rarely in the thymus. The efficiency of precursor units of neonatal mice when they become functional approximates that of the mature animal when based on the doubling time of plaque-forming cells (PFC). Possible explanations of the initial appearance of incomplete precursor units have been discussed.