Long-lasting skin allograft tolerance in adult mice induced across fully allogeneic (multimajor H-2 plus multiminor histocompatibility) antigen barriers by a tolerance-inducing method using cyclophosphamide.

A new method of cyclophosphamide (CP)-induced skin allograft tolerance in mice that can regularly overcome fully allogeneic (major H-2 plus non-H-2) antigen barriers in mice has been established. The components of the method are intravenous or intraperitoneal administration of 50-100 micrograms of anti-Thy-1.2 mAb on day -1, intravenous injection of 90 x 10(6) allogeneic spleen cells mixed with 30 x 10(6) allogeneic bone marrow cells from the same donor on day 0, and intraperitoneal injection of 200 mg/kg CP on day 2. In each of four fully allogeneic donor----recipient combinations, including C3H/HeJ (C3H; H-2k)----C57BL/6J(B6; H-2b), B6----C3H, BALB/cByJ (BALB; H-2d)----B6, and BALB----C3H, long-lasting survival of skin allografts was induced in most of the recipient mice. The specific tolerant state induced was dependent on the doses of the antibody and bone marrow cells used. The optimal timing of CP treatment to induce tolerance was found to be 1-3 d after the stimulating cell injection. Treatment with the anti-Thy-1.2 antibody together with CP on day 2 after the cell injection on day 0 also induced profound tolerance. In the B6 mice made tolerant of C3H with antibody, C3H spleen cells plus C3H bone marrow cells, and then CP, a minimal degree of stable mixed chimerism was established and the antitolerogen (C3H) immune responses examined here, including delayed footpad reaction (DFR), CTL activity, and capacity for antibody production against donor-strain antigens were abrogated in a tolerogen-specific manner. From cell transfer experiments, the mechanism of tolerance could be largely attributed to reduction of effector T cells reactive against the tolerogen, and strong suppressive influences that might prolong skin allograft survival directly were not detected in the tolerant mice. Moreover, pretreatment with anti-Thy-1.2 antibody or anti-L3T4 (CD4) antibody was more effective than pretreatment with anti-Lyt-1 (CD5) antibody or anti-Lyt-2 (CD8) antibody as an initial step in tolerance induction. These results suggest that permanent tolerance to fully allogeneic skin grafts may be induced because antibody given before the stimulating cell injection reduces the number of reactive T cells in the recipient mice. This antibody treatment may facilitate an antigen-stimulated destruction of responding and thus proliferating cells with CP by preventing a possibly less proliferative, more rapid maturation of reactive T cells or by destroying residual effector T cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Humoral and cell-mediated immune responses in fully allogeneic bone marrow chimera in mice.

AKR mice were protected from lethal irradiation and established as long-lived chimeras by transplanting allogeneic C57BL/6 (B6) bone marrow that had been treated in vitro with anti-Thy-1 antiserum without complement. In these chimeras, which were designated [B6 {arrow} AKR], virtually all the thymus and spleen cells were shown to be derived from the B6 donor; several immune functions studied in these chimeras were as follows: (a) The chimeric mice were tolerant of histocompatibility antigens of both donor and recipient strain and nearly fully reactive to antigens of third party, as revealed by Simonsen's splenomegaly assay. The tolerance of these chimeras could not be attributed to suppressor cells but was compatible with clonal depletion. (b) Proliferative responses to concanavalin A, phytohemagglutinin, and lipopolysaccharide as well as natural killer and antibody-dependent cell- mediated cytotoxicity activity of the chimeric mice was normal. (c) Plaque- forming cell (PFC) assays of antibody responses to sheep erythrocytes (SRBC) showed gross deficiency in the primary response of the [B6 {arrow} AKR] and [AKR {arrow} B6] chimeras. By contrast, [B6-H-2(k)(E(k)) {arrow} AKR] H-2-compatible chimeras and [AKR {arrow} AKR] syngeneic marrow transplanted mice had normal primary PFC responses. PFC responses after secondary stimulation with SRBC, however, revealed vigorous direct plaque formation and substantial but somewhat smaller indirect plaque formation in the [B6 {arrow} AKR] chimeras. This observation favors operationally the concept of adaptive differentiation proposed by Katz et al. (44). (d) Analysis of ability of the chimeras to develop and express delayed-type hypersensitivity responses to contact sensitizer (2,4-dinitro-l-fluorobenzene [DNFB]) showed no apparent immunodeficiency of either chimeras to this form of immunization. Development of immunologic tolerance to DNFB, however, was grossly deficient in [B6 {arrow} AKR] chimeras but normal in [AKR {arrow} AKR], [B6 {arrow} B6], and [E(k) {arrow} AKR] chimeras. These findings indicate that full chimeras across major histocompatibility complex have considerable immunologic vigor even though primary immune responses that require histocompatibility between interacting cell types are initially defective.

Prevention of the localized and generalized Shwartzman reactions by an anticomplementary agent, cobra venom factor.

Both localized and generalized Shwartzman reactions were induced in the same rabbits by simultaneous administration of preparatory intravenous and intradermal doses of endotoxin followed in 24 hr by the provocative dose. Control rabbits with more than 80% positive responses showed corresponding changes of platelet, white blood cell, fibrinogen, and hemolytic complement levels. Circulating fibrinogen and fibrin degradation products were detected shortly after the preparatory dose and persisted for at least 3 days. Rabbits given cobra venom anticomplementary factor showed hypocomplementemia (less than 10% of normal), leukocytosis, and elevated fibrinogen levels. After the administration of endotoxin, only one of 15 CVF-treated animals developed a Shwartzman reaction and that was mild. These rabbits showed only minor changes of platelet and fibrinogen levels throughout the experiment although their white blood cell responses were similar to those of the control group. No detectable fibrinogen and fibrin degradation products appeared in circulation, and the hemolytic complement activity increased gradually beginning with the preparatory dose of endotoxin. Thus depletion of terminal complement components (mainly C3) in rabbits is protective against the development of both localized and generalized Shwartzman reactions; its mechanism of action is probably through the sparing of platelets by inhibiting platelet-endotoxin interaction. The essential role of the complement system in Shwartzman reaction indicates that this coagulopathy probably represents a manifestation of immunologic injury.

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 10(6)) 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 10(6)), 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 (F(1) 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 thymus function. II. Cooperative effect of newborn and embryonic hemopoietic liver cells with thymus function.

Significant immunological restoration of 45-day old, neonatally thymectomized C3Hf mice was obtained by the cooperation of syngeneic newborn or embryonic hemopoietic liver cells with thymic function. Thymic function or cells alone are almost ineffective or restore approximately 10% of the animals. Newborn liver cells are effective in association with thymus grafts or humoral thymic function (thymoma grafts and thymus or thymomas in diffusion chambers). Embryonic liver cells are ineffective, even in large numbers, when associated with humoral thymic function. On the other hand, embryonic liver cells are effective in the cooperative effect only in association with viable thymus grafts, preferably syngeneic, whether the grafts were placed subcutaneously, intraperitoneally, or under the kidney capsule. Dispersed viable thymic cells are ineffective in association with embryonic liver cells. Cells capable of cooperating with humoral thymic function start to appear to embryonic liver by day 19-21 of gestation and are detectable until day 5-6 postbirth. Embryonic hemopoietic liver cells from 12 to 18 days of gestation contain cells capable of cooperation only with viable free thymus grafts and not with humoral thymic function. A prethymic cell population of partially differentiated cells of hemopoietic origin, insensitive to humoral activity of the thymus but requiring thymic stroma and traffic through the thymus is postulated to explain our results. This population of prethymic cells can become postthymic through this process and eventually develop into competent cells. Postthymic cells are characterized by their sensitivity to humoral activity of the thymus and by their wide distribution in the lymphohemopoietic tissues of newborn and young adult mice.

Carcinogen-induced tumors of the thymus. IV. Humoral influences of normal thymus and functional thymomas and influence of posthymectomy period on restoration.

A progressive decrease of the restoring effectivity of syngeneic or allogeneic thymus and functional thymoma grafts was observed when the treatment of neonatally thymectomized mice was delayed. Early treatment (5-20 days postthymectomy) was effective, while the number of restored animals was markedly decreased after late treatment (30-50 days postthymectomy). Similar results were obtained with subcutaneous or intraperitoneal thymus grafts and with thymus grafts within cell-impenetrable diffusion chambers. After the onset of the postthymectomy-wasting syndrome the only successful treatment was the implantation of multiple thymus grafts. On the other hand, single thymus grafts, thymoma grafts, or thymus or thymoma within diffusion chambers were ineffective. When spleen cells from 5-day old or 45-day old neonatally thymectomized animals were given in association with thymoma grafts, only the cells derived from the 5-day old thymectomized mice proved effective in restoring wasted thymectomized hosts. These results suggest that a population of cells sensitive to the action of the thymus decreases progressively with time in the absence of thymic function.

Ontogeny of bursal function in chicken. 3. Immunocompletent cell for humoral immunity.

To study the occurrence of immunocompetent cells directly responsible for antibody production, cells from yolk sac, embryonic liver, bursa of Fabricius, bone marrow, spleen, or thymus were injected together with SRBC and Brucella abortus into 4-day old cyclophosphamide-treated chicks. A second stimulation was given 4 days later, and samples taken 4 days thereafter were used for antibody titrations. During ontogeny, immunocompetent cells appeared in significant numbers first in the spleen for anti-SRBC responses, but in the bursa for anti-Brucella responses. Later these cells were also found in thymus and bone marrow. In the bursa, cells immunocompetent for anti-SRBC response were not encountered in significant numbers. The slight response to SRBC by transferred bursa cells reflects the presence of stem cells and their immediate descendents in the bursa at different stages of development. These findings are compared with the development and maturation of the stem cell responsible for humoral immunity. In the bursa, development of the stem cell population precedes that of immunocompetent cells. The opposite relationship was found in bone marrow, spleen, and thymus where immunocompetent cells were always present some weeks before the appearance of cells capable of achieving a long-lasting reconstitution of bursa-dependent functions. These observations reveal that the bursa seeds out immunocompetent cells during its entire postembryonic development, but does not release the lymphoid stem cell population before this population has matured sufficiently and before the bursa itself, after fulfilling its function, starts to involute.

Studies on thymus function. 3. Duration of thymic function.

The immune functions of neonatally thymectomized C3Hf mice exposed only temporarily to thymus function show a progressive decay with time in the absence of the thymus. The immune responses studied at different ages in the range of 100-600 days were: first-set rejection of H-2-compatible and incompatible skin allografts, second-set rejection of skin allografts, capacity of spleen cells to produce graft-versus-host reactions in F(1) hybrids, resistance to infection with mouse hepatitis virus, and response of spleen cells to phytohemagglutinin in vitro. These long-term studies had the purpose of determining the duration of the restoration induced by thymus function when the mice were exposed only temporarily to it. Different models were used but the two basic ones were: (a) mice grafted intraperitoneally at 15 days of age with a syngeneic thymus that was removed surgically at 10, 20, or 30 days after grafting, and (b) mice grafted at 15 days of age with allogeneic strain A thymoma or C57BL thymus, these representing situations in which there is spontaneous rejection of the restoring graft. In all the experimental models used, the animals were restored when tested at 100 days of age, but progressively became immunologically incapacitated at 200-300 days of age. From the more controlled experiments in which the restoring thymus graft was removed surgically, the following conclusions can be drawn. (a) A short exposure to a thymus graft can produce restoration of immune functions in neonatally thymectomized mice, but this restoration is not self-sustaining in the absence of the thymus and declines progressively with age. The decline usually starts at 200-300 days of age. (b) This was especially clear in experiments in which the same animal was tested twice in its lifetime for capacity to produce graft-versus-host reactions; these animals were competent at 100 days and became incompetent at 400 days of age. (c) The shortest period of thymic exposure studied was 10 days; if vascularization of the graft is taken into account, 2-3 days of thymic function are sufficient to produce restoration. (d) The immune decay observed in the thymectomized animals exposed temporarily to thymus was more profound than the physiological decay of immunity observed in control animals of similar age. (e) Of all the tests studied, the response of spleen cells to phytohemagglutinin was to be preserved the longest in animals exposed only temporarily to thymic function. The present results were interpreted in accordance with our previous findings indicating that a population of postthymic cells can be developed by temporary exposure of neonatally thymectomized animals to thymic function, but that this population is not self-sustaining in the absence of thymus and progressively decays by physiological attrition.

Development of chicken lymphoid system. I. Synthesis and secretion of immunoglobulins by chicken lymphoid cells.

Synthesis and secretion of Ig by chicken lymphoid cells was studied. Both spleen and bursa cells synthesize and secrete IgM and IgG whereas Ig was not detected in thymus cells. In contrast to the spleen cells which synthesize H and L chains in balanced quantities, the bursa cells synthesize and secrete free L chains. In addition to the lymphoid cells which secrete IgM or IgG, the bursa appears to contain a cell population which synthesizes nonsecretory Ig. The structure of this Ig was studied by specific serological precipitation and by SDS-acrylamide gel electrophoresis. The H chains of this nonsecretory Ig are serologically related to micro-chains and exhibit a smaller molecular weight (i.e., approximately 50,000) in SDS-acrylamide gel electrophoresis than H chains of IgG and IgM synthesized by the spleen cells (i.e., approximately 70,000).

Development of chicken lymphoid system. II. Synthesis of primordial immunoglobulin M by the bursa cells of chick embryo.

Development of Ig-synthesizing cells in the bursa of chick embryo was studied by immunohistochemical staining method as well as by in vitro incorporation of leucine-(3)H into Ig. Ig-synthesizing cells are first detected in the bursa of a 14 day old chick embryo and increase with the maturation of the embryo. Acrylamide gel analysis of leucine-(3)H-labeled Ig shows that synthesis of nonsecretory IgM-H(0) precedes that of secretory IgM-H, reflecting an ontogenetic sequence of development of lymphoid cells synthesizing IgM. Since IgM-H(0) is not secreted, we further studied biochemical differences between two heavy chains. The difference is attributable to lack of galactose attachment to H(0) chains. It is proposed that during differentiation of lymphoid cells synthesizing and secreting Ig, attachment of galactose may play an obligatory role in the development of cellular capacity for Ig secretion.

Quantitative effects of nutritional essential amino acid deficiency upon immune responses to tumors in mice.

Mice were fed diets deficient in a single essential amino acid, and the primary immune responses to inoculation of allogenic tumor cells was measured by in vitro assay of cellular immunity. Moderate reduction of the amino acids phenylalanine-tyrosine, valine, threonine, methionine-cystine, isoleucine, and tryptophane in the diet produced profound depression of hemagglutinating and blocking antibody responses, although cytotoxic cell-mediated immunity remained intact. These diets had previously been shown to result in a selective depression of tumor growth in mice. Limitation of the amino acids arginine, histidine, and lysine in the diets gave rise to only slight depression of the immune responses. These diets had previously been shown to produce a proportional decrease in both tumor growth and host body weight. Moderate leucine restriction resulted in a paradoxical depression of cytotoxic cell-mediated immunity with little effect on serum blocking activity. Slight increases had previously been noted in the weight of tumors in mice fed leucine-restricted diets. Deficiency or imbalance of essential amino acids in the diet may produce profound depression of immune responses and apparent, marked changes in the immune resistance of the host animal to tumors.

Morphological and functional studies of fetal thymus transplants in mice.

The fetal thymus at 13 days of gestation withstands transplantation and develops normally under the renal capsule of a syngenic host. Distinct differences were observed between the fetal thymus grafts and grafts from neonatal or adult thymus donors. The fetal thymus graft did not undergo the rapid and severe necrosis observed when adult thymus was grafted. Furthermore, when thymuses were transplanted into allogenic recipients, rejection was delayed. The fetal thymus was as effective as the adult thymus in restoring syngenic neonatally thymectomized mice and far superior to adult thymus when grafted into allogenic recipients. These observations seem relevant to clinical efforts to restore immunocompetence in patients with congenital absence of the thymus.

Suppressor cell activity after concanavalin A treatment of lymphocytes from normal donors.

Pretreatment of normal human peripheral blood lymphocytes with the plant lectin, concanavalin A (Con A), results in inhibition of blast transformation and [3H]thymidine incorporation by untreated allogeneic lymphocytes from healthy volunteers donors in one-way mixed leukocyte culture. Similarly, responses to mitogens, certain microbial antigens, and allogeneic lymphocytes are inhibited by Con A-treated allogeneic cells. Con A pretreated autologous lymphocytes can also be induced to manifest suppressor activities. This antimitotic effect occurs without evidence of cytotoxicity and is active on de novo lymphocyte responses and does not require prior sensitization of the cells being tested. Suppression of the lymphocyte response to pokeweed mitogen, a potent B-cell stimulator, by Con A-pretreated suppressor cells was not as consistent as was inhibition of response to other mitogens, including phytohemagglutinin and Con A. Furthermore, suppression of lymphocyte transformation to the microbial antigens, tuberculin purified protein derivative, and Canadida albicans extracts could be similarly induced by Con A pretreatment of either allogeneic or autologous cells. Induction of autologous suppressor activity in lymphocytes from healthy donors is compatible with a model that includes a role for suppressor cells in the modulation of the normal immune response.

Escape from isoantiserum inhibition of lymphocyte-mediated cytotoxicity.

Isoantiserum (IS) inhibition of lymphocyte-mediated cytotoxicity (LMC) was studied using an in vitro (51)Cr release assay system. In the early phase of incubation, LMC was competitively inhibited by IS. However, as the incubation continued, LMC irreversibly overcame IS inhibition (the "escape" phenomenon). Addition of fresh antiserum did not alter the course of the escape. Low-temperature incubation of isoantibody-coated target cells delayed the onset of the escape. We have excluded the possibility that the escape phenomenon is induced by complement or by LMC mediated by antigen-antibody complex. It is hypothesized that antibody directed toward an actively metabolizing target cell induces an alteration in the cell membrane that alters further interaction with the antibody. However, sensitivity to lymphocyte cytotoxicity is maintained.

Pre-B cells and other possible precursor lymphoid cell lines derived from patients with X-linked agammaglobulinemia.

A group of unique Epstein-Barr virus-containing cell lines was derived from the bone marrow of three patients with X-linked agammaglobulinemia. Efforts to obtain cell lines from the peripheral blood of these patients were uniformly unsuccessful. Immunofluorescence analyses as well as biosynthetic studies with [(35)S]methionine indicated unusual patterns of Ig synthesis in many of these bone marrow derived lines. Seven of the lines were of particular interest in that two produced no Ig of any type; two others showed no Ig by fluorescence but small amounts by [(35)S]methionine labeling; one expressed only cytoplasmic mu chains without any evidence of light chain synthesis, and two produced primarily mu chains with only slight amounts of light chains. One of the lines without membrane or cytoplasmic Ig studied in detail grew like a typical lymphoid line and was carried in intermittent culture over a period of 2 yr without Ig expression. One line grew quite differently and resembled the round cell type described previously, which has been obtained from a variety of sources. The cell line with cytoplasmic mu chains and no light-chain expression had the characteristic properties of pre-B cells. Three normal type Ig-producing cell lines also were obtained from the patients. The accumulated evidence obtained in the present study indicates that these unusual cell lines represent normal precursor cells of the B-cell lineage; these grew out in these cases because of the virtual absence of mature B cells that ordinarily overgrow the culture system. However, the possibility that in certain instances they reflect abnormal Ig synthesis characteristic of the disease has not been ruled out.

Identification of a p69,71 complex expressed on human T cells sharing determinants with B-type chronic lymphatic leukemic cells.

In the course of generating monoclonal antibodies to human thymus-dependent differentiation antigens, we were able to define specificities shared by T cells and by cells from patients with chronic lymphatic leukemia that were not detectable on normal B cells. In particular, one of these antibodies was reactive by indirect immunofluorescence with greater than 95% of the thymocytes and 80--95% of nonadherent sheep erythrocyte-rosetting peripheral blood lymphocytes (PBL), but was unreactive with normal B cells or cell lines derived from PBL by Epstein-Barr virus transformation. However, the leukemic cells from 11 of 14 patients with B-type chronic lymphatic leukemia were found to express detectable concentrations of this surface determinant. The target antigen recognized by this monoclonal antibody was shown by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be a p69,71 complex. Our findings suggest a possible relationship between this antigen and the previously described GIX system in the mouse.

Postthymectomy wasting associated with autoimmune phenomena. I. Antiglobulin-positive anemia in A and C57BL-6 Ks mice.

1. Mice of A and C(57)BL/6 Ks strains, thymectomized at birth acquire wasting disease in 84.1% (A) and 77.1% (C(57)BL/6 Ks) of the cases. There is no sex predelection. 2. Anemia in these animals is characterized by shortened red cell survival and increased fragility to hypotonic salt solutions. Among thymectomized A mice reticulocytosis is absent and extramedullary hematopoiesis is found in the spleen in the presence of bone marrow hypoplasia for the erythroid and lymphocyte series. 3. Positive antiglobulin tests of the red cells were observed in all the thymectomized C(57)BL/6 Ks (7/7) and 71.2% of the A strains (62/87). Normal mice do not show positive Coombs' tests. 4. The globulin coat on the A strain consists of IgM, whereas beta(1C) and IgG are not detectable. By contrast, red cell coats of NZB mice developing spontaneous autoimmune hemolytic anemia show IgM and beta(1C), but these erythrocytes do not react with anti-gamma chain antibodies. Another difference in the globulin coats of the two types of erythrocytes is that the IgM on NZB red cells has available light chain determinants but these are apparently hidden or absent in the case of sensitized erythrocytes. The difference in antibody coating, association with a component of complement in one but not the other, suggests a different mechanism for the immune surface phenomenon in each instance. 5. Anemia in NZB mice is associated with reticulocytosis while that in thymectomized A mice is not. 6. Thymectomy appears to initiate a chain of events leading to a series of autoimmune phenomena which may be due to alteration in host response consequent to loss of thymic tissue and thymic dependent functions or alternatively to infection to which increased susceptibility exists as a result of thymic extirpation.

The functions of the thymus system and the bursa system in the chicken.

The bursa of Fabricius and the thymus are "central lymphoid organs" in the chicken, essential to the ontogenetic development of adaptive immunity in that species. Surgical removal of one or both of these organs in the newly hatched chicken, followed by sublethal X-irradiation the next day, has permitted recognition of two morphologically distinct cell systems in the "peripheral lymphoid tissues" of the spleen, gut, and other organs, and clear definition of the separate functions of each cell system. The thymus-dependent development is represented morphologically by the small lymphocytes of the circulation and the white pulp type of development in the tissues. As in mammals, the thymus-dependent tissues of the chicken are basic to the ontogenesis of cellular immunity: graft versus host reactions, responses of delayed hypersensitivity and homograft rejection; and play a less clearly defined role in the antibody response to at least some antigens. Thymectomized-irradiated chickens are deficient in all these responses, and grow more slowly than any of the other experimental groups. In these animals germinal centers, plasma cells, and capacity for immunoglobulin synthesis remain intact. The bursa-dependent development is represented morphologically by the larger lymphocytes of the germinal centers and the plasma cells, and functionally by the immunoglobulins. Bursectomized-irradiated chickens are agammaglobulinemic and unable to produce detectable antibody despite intense, repeated stimulation with bovine serum albumin and Brucella abortus organisms. The thymus-dependent development in these animals seems to be normal; they have adequate numbers of lymphocytes in the circulation and tissues, are able to reject skin homografts, though more slowly than usual, and to exercise graft versus host reactions. The short life span of these chickens has precluded adequate study of responses of delayed hypersensitivity. There was no evidence of significant impairment of reticuloendothelial function in either the bursectomized-irradiated or the thymectomized-irradiated group, as judged by the clearance of colloidal gold and I(131)-tagged keyhole limpet hemocyanin.

The IgA system. I. Studies of the transport and immunochemistry of IgA in the saliva.

1. Five patients with congenital or acquired agammaglobulinemia, lacking detectable IgA in serum or saliva, were transfused with 1 to 2 liters of normal plasma. In 2 of these patients IgA was demonstrated in parotid saliva collected after transfusion, but in none of the 5 was salivary IgG or IgM found. This observation indicates the selective transport of IgA into saliva. 2. The observation by others of an immunochemical difference between serum and sahvary IgA globulin was confirmed. In contrast to serum IgA, salivary IgA is attached to a protein having antigenicity which migrates as a gamma(1) globulin. We have termed this protein component "transport piece". 3. The transport piece has been found in an unbound form in the saliva of persons completely lacking IgA: agammaglobulinemic patients, ataxia-telangiectasia patients, a healthy person lacking IgA, and a newborn infant. Free transport piece still occurs in the normal child's saliva after IgA production begins. By adulthood there is usually no free transport piece in the saliva. 4. Heat-aggregated salivary IgA, like heat-aggregated serum IgA, does not fix complement. 5. Our findings offer support for the view that there is a distinct local antibody system for the protection of the mucous surfaces.

Localization of immunological complexes fixing beta1C (C3) in germinal centers of lymph nodes.

28 human and 60 experimentally stimulated rabbit lymph nodes were studied by means of light microscopy and immunofluorescence. 21 of the 28 human lymph nodes showed well-developed germinal centers. IgM, IgG, and the beta(1C) component of complement were found in the same distribution within germinal centers when examined in serial cryostat sections. 36 rabbits were stimulated with Brucella antigen, and 24 rabbits with BSA. A strikingly consistent correlation between distribution and appearance of specific staining for rabbit beta(1C), IgM, and IgG was observed; when lymph nodes were stimulated with BSA, antigen and specific antibody were present. Treatment of unfixed sections with citrate-buffered saline at low pH resulted in complete elution of immunoglobulins, beta(1C), and BSA from rabbit germinal centers, and in marked diminution of IgG and IgM in human germinal centers, while at the same time plasma cells remained strongly fluorescent. Specific selective fixation of heterologous (human) complement in rabbit germinal centers positive for beta(1C), IgG, IgM, and BSA was also obtained. These data present strong evidence for the existence within germinal centers of antigen-antibody complexes which fix at least the beta(1C) component of complement in vivo. The possibility of complete elution of immunoglobulins from rabbit germinal centers can be taken as evidence that, at least for 20 days after primary and secondary stimulation, a major component of the immunoglobulins present in germinal centers is not produced locally but accumulates at the surface of cells.