Changes produced by pertussis antigen on the blood cells and lympho-haemapoietic tissues after early and late thymectomy or splenectomy

The effect of thymectomy and splenectomy in C3H/Bi mice on the responses of circulating leucocytes and on morphological changes of the haematopoietic tissues after injection of pertussis vaccine has been studied.

After pertussis all mice showed depletion of lymphoid cells in all the lymphoid organs as well as in bone-marrow and an increased number of leucocytes, lymphocytes, neutrophils and monocytes in the circulation. Neonatal thymectomy decreased lymphocytosis produced by pertussis. Thymectomy, at all ages studied, fostered an increase in the number of monocytes and polymorphonuclears in circulation. Splenectomy at birth or early in life provoked an increase in levels of circulating polymorphonuclears and lymphocytes in pertussis treated animals.

In neonatally thymectomized mice the depletion of lymphoid cells from lymphoid tissues after pertussis could be shown to include the thymic-independent areas. The depletion of small lymphocytes from thymus following pertussis persisted longer than depletion of small lymphocytes from spleen, marrow or lymph nodes. The longer persistence of lymphoid depletion in the thymus than in peripheral lymphoid tissues is, we believe, to be related to the central lymphoid function of thymus as a site of differentiation of lymphoid cells and to the aloofness of thymus from recirculation of fully differentiated peripheral lymphocytes.

     

Local Graft-Versus-Host-Reaction in Mice Specifically Inhibited by Anti-Receptor Antibodies

The local graft-versus-host (GVH) reaction provoked by parental spleen cells in F1 mice was shown to be T-cell-dependent. GVH reactions were suppressed in F1 hybrid mice immunized with parental T lymphocytes of the same genotype, but not in F1 mice immunized with parental B cells. In some cases this immunity could be passively transferred by serum into normal F1 mice. The specific activity of such sera could be removed by absorption with either parental T or B cells. Some of the F1 antisera were specificlly cytotoxic for parental GVH-reactive lymphocytes.     

The development of lymphocytes with T- or B-membrane determinants in the human foetus

The development of T- or B-membrane determinants on human foetal lymphoid cells was studied by the direct immunofluorescence technique, using a tetramethyl rhodamine isothiocyanate (TRITC) labelled horse antihuman T-cell conjugate (ATC) for the detection of T lymphocytes and a fluorescein isothiocyanate (FITC) labelled goat antihuman Fab conjugate for the demonstration of Ig-bearing B lymphocytes. Human foetal lymphocytes were also tested for spontaneous rosette formation with sheep red blood cells (SRBC).

Cell suspensions of liver, spleen, thymus, bone marrow and blood of twenty-five human foetuses of 5·5–26 weeks of gestational age have been investigated. ATC-positive lymphoid cells were first seen in the liver at 5·5 weeks; E rosette-forming cells (ERFC) and Ig-bearing lymphoid cells were first found at 9 weeks. ERFC were also present in the thymus at 9 weeks. By 12 weeks, fluorescent B and T lymphocytes were found in bone marrow and spleen. ERFC were also found in bone marrow at this age, but not in spleen. At 15 weeks, more than 80% of blood lymphoid cells had T or B determinants.

A difference in the reactivity of lymphoid cells with the ATC and their capacity to form E rosettes was observed. In liver and spleen, the ATC determinant was detectable before the SRBC receptor. In bone marrow, blood and thymus the ATC determinant was found on a higher percentage of lymphoid cells than was the SRBC receptor when those organs were first investigated. During the entire investigated period of gestation, the majority of lymphoid cells in liver and bone marrow did not react with either of the conjugates, nor did they form E rosettes. In all organs investigated, except in the thymus, lymphoid cells were occasionally seen which reacted with both conjugates. By the 16th week of foetal age, more than 90% of lymphoid cells in thymus, spleen and blood had acquired T- or B-membrane determinants.

     

Hormones and immunological capacity: II. Reconstitution of antibody production in hormonally deficient mice by somatotropic hormone, thyrotropic hormone and thyroxin

Hypopituitary dwarf mice are immunologically deficient. This deficiency can be overcome by injection of somatotropic hormone and thyroxin. Antibody formation in hormonally reconstituted mice as measured by the number of plaque-forming cells against sheep erythrocytes equals or surpasses that of normal mice. The number of nucleated spleen cells is increased in both normal and dward mice after treatment with hormones. The hypotrophic thymus and peripheral lymphoid tissue of dwarf mice can be reconstituted to normal by treatment with somatotropic hormone and thyroxin.

Anti-somatotropic hormone and anti-thyrotropic hormone antisera produce suppression of antibody formation. These effects can be reversed by somatotropic hormone and thyrotropic hormone. The anti-hormone antisera produce an involution of thymus and other lymphatic organs. A parallelism exists between involution of the lymphoid tissue, neutralization of circulating somatotropic hormone and depression of antibody production.

These results stress the importance of the thymus—hypophysis relationship for cell differentiation with particular reference to the maturation of the immunological capacity.

     

Quantitative analysis of the chimæric state in mice: II. Cytological examination of the proportion of proliferating donor and host cells in runt disease in mice

The distribution of donor cells in the lymphoid organs of mice suffering from acute runt disease was investigated by means of the cytological marker (T6). In all the strain combinations studied relatively low proportions of donor cells were identified in the spleen and bone marrow. In the A→CBA-T6T6 and C57BL→CBA-T6T6 combinations high proportions of donor cells were found in the lymph nodes and thymus. Few donor cells at all lymphoid sites were identified in the CBA-T6T6→C57BL combination, though the recipients showed unquestionable clinico-pathological signs of runt disease.

Despite marked splenomegaly in 12-day-old runts, the mean total number of cells in the spleen was similar to that in normal mice and mice injected at birth with isologous cells. In the A→CBA-T6T6 and C57BL→CBA-T6T6 combinations donor cells significantly contributed to the total cellularity of the enlarged spleen, in contrast to the CBA-T6T6→C57BL combination in which the host's spleen consisted almost exclusively of host cells.

Grafting tests showed that runted mice can be either non-specifically or specifically tolerant which seemed to depend on the degree of lymphoid atrophy and the degree of chimerism. Some of the animals that eventually rejected skin grafts from spleen inoculum donors, continued to be chimeric for donor lymphoid cells.

A mechanism for runt disease could be mutual interaction between donor and host cell populations causing reduction of the host's lymphoid reserve and consequent appearance of clinical symptoms of disease.

     

The lymphoid tissues in mice with congenital aplasia of the thymus

The lymph nodes, spleens and Peyer's patches from seven mutant nu nu mice with agenesis of the thymus were compared with similar tissues from eight phenotypically normal littermate controls.

In all lymph nodes examined from the nu nu mice the thymus-dependent areas were virtually `empty' of lymphocytes. In the corresponding areas in the spleen and Peyer's patches the numbers of lymphocytes were also significantly reduced.

The nu nu mice are potentially important as experimental tools in delineating the role of the thymus in the development of the lymphoid system, and as an experimental model of thymic alymphoplasia in the human.

     

Studies on the immunosuppresive properties of asparaginase

The effect of asparaginase on lymphocyte kinetics and on immunological competence of normal and adrenalectomized mice has been studied. Treatment of mice with this enzyme results in lymphocytopenia and atrophy of lymph nodes, thymus and spleen. The lymph node lymphocytes from asparaginase-treated mice do not migrate normally to lymphoid organs when injected intravenously into syngeneic recipients. Splenic lymphocytes from asparaginase-treated mice induce significantly less graft-versus-host reaction when injected into F₁ hybrids and incorporate significantly less thymidine into DNA when cultured in vitro with phytohaemagglutinin than do cells from normal mice. Skin graft rejection and the formation of antibody to sheep erythrocytes is significantly inhibited by administration of asparaginase. The immunosuppressive actions of asparaginase are manifested in adrenalectomized mice. Studies on the inhibition of phytohaemagglutinin-induced lymphocyte transformation by asparaginase suggest that asparagine depletion underlies the effect of asparaginase on lymphocytes.     

Effect of allogeneic cell interaction on the primary immune response in vitro. Cell types involved in suppression and stimulation of antibody synthesis

A graft-versus-host (GVH) reaction was induced in F1 hybrid mice by the inoculation of spleen cells from one of the parental strains. One week later the spleen cells from the recipients were cultured during the conditions for obtaining a primary immune response in vitro described by Mishell & Dutton (1967). It was found that the antibody response against the thymus-dependent antigen sheep red cells (SRC), as well as the thymus-independent antigen lipopolysaccharide from Escherichia coli 055:B5 (CPS) was markedly depressed. Spleen cells from mice subjected to a GVH reaction (GVH cells) also inhibited the antibody response of normal cells in vitro. The inhibitory effect of the GVH cells on normal cells was not sensitive to treatment with anti-θ serum, but could be completely abolished by treatment with iron powder, which removes adherent cells.

By culturing cells of two different mouse strains together in vitro it was possible to obtain stimulation or inhibition of the antibody response depending on the total cell number per dish.

The relation of these results to the phenomenon of antigenic competition is discussed. It is suggested that antigenic competition is caused by a non-specific inhibition of cell proliferation, possibly mediated by a locally acting factor. Both thymus-derived and bone marrow-derived cell lymphocytes are affected by the phenomenon. The cells initially responsible for the inhibition seem to be antigen-activated thymus-derived cells (T-cells), which by secondarily activated cells such as macrophages inhibit other cells. A GVH reaction, which generally leads to antigenic competition may, when less pronounced, cause stimulation of the antibody response.

     

Cells involved in the graft-versus host reaction in vitro

The cell types involved in the cellular immune response were studied with the GVH in vitro as a test system. Comparison of the activities of cells of different lymphoid organs in the GVH in vitro showed the cells of peripheral lymph glands and thoracic duct to be the most active. Second in activity were the cells from peritoneal exudate, mesenterial lymph nodes and spleen. Weak activity was found in thymus and bone marrow cells, whereas cells from Peyer's patches showed no activity at all. Treatment of donor animals with ATS, cortisone or cyclophosphamide simultaneously with (acceptor-) transplantation antigen, resulted in spleen cells which were less active in the GVH in vitro. No such results were obtained after treatment of donor animals with dimethylbenzanthracene or cyclophosphamide without antigen. Fractionation of donor spleen cells on a bovine serum albumin density gradient resulted in an accumulation of activity in one of the high density fractions (D), whereas this was reduced in the low density fractions (C, B and A) and absent in the highest density fraction (E). Separation of thymus cells on a glass bead column coated with transplantation antigen resulted in augmented activity of the adherent cell fractions, while the effluent cells were less active than the original cells. Fractionation of cells on an uncoated column resulted in adherent and non-adherent cells which were both inactive in the GVH in vitro.     

Reduced Capacity to Produce Specific 'Effector' Cells after Injection of CBA Mice with C3H Cells

Lymphocytes from mice of strain CBA are strongly MLC-responsive to lymphocytes from the H-2 compatible- but M-antigen-incompatible strain C3H. This strong reactivity disappears after infusion of CBA mice with C3H lymphocytes. This study shows that the host-versus-graft reactivity (swelling of local lymph node after antigen injection) is specifically reduced after injection of CBA mice with C3H × CBA spleen cells However, lymphocytes from such mice showed a specifically increased GVH reactivity (inhibition of erythroid cell growth) compared with lymphocytes from unimmunized mice. Lymphocytes from normal CBA mice showed a high proliferative rate in the spleens of irradiated C3H × CBA mice. Such 'educated' cells showed strongly increased specific GVH reactivity. Lymphocytes from CBA mice previously injected with C3H×CBA cells showed reduced capacity to proliferate when injected into irradiated C3H × CBA hybrids and a poor capacity to develop new 'effector' cells reactive against C3H × CBA bone marrow target cells. The results indicate that the presence of specifically 'MLC responsive' lymphocytes in a lymphoid cell population is a prerequisite of its production of 'effector' cells able to respond in this GVH assay     

Effects of Anti-T Cell (Theta) and Anti-B Cell (Beta) Serum on the Immune Response in Mice

Heterologous anti-B cell (anti-beta) serum was prepared in rabbits against the spleen from neonatally thymectomized mice. The anti-beta serum, after absorption with thymus, is cytotoxic for bone marrow, bone marrow-derived cells, fetal liver and peritoneal lymphocytes. The cytotoxicity to the B cell can be absorbed out with bone marrow.

The cytotoxic effects of anti-beta serum on spleen and lymph node cells is compared to that of anti-theta serum. The data suggest that spleen has relatively more B than T cells, while lymph node has relatively more theta-positive cells.

To test the effect of anti-beta and anti-theta serum on the functional activity of lymphoid cells, C57 spleen or thymus was pre-incubated with the antiserum, in the presence of complement, and tested in vivo for graft-vs-host activity or transfer of an adoptive immune response to SRBC.

Treatment with anti-beta serum does not decrease the graft-vs-host activity of thymus or spleen cells. Anti-theta serum does decrease the graft-vs-host activity of both thymus and spleen cells.

Neither anti-beta serum nor anti-theta serum affect the phagocytic activity of peritoneal macrophages.

Both anti-beta serum and anti-theta serum decrease the transfer of an adoptive primary and secondary immune response to SRBC. A combination of anti-theta and anti-beta treated spleen can transfer adoptive immunity. Thymus and bone marrow can reconstitute the immunocompetence of anti-theta or anti-beta treated spleen respectively.

The results suggest that T cells alone can mount a graft-vs-host reaction and that this activity is not affected by anti-beta serum. The transfer of a humoral antibody response, on the other hand, requires functionally active T- and B-cells. This holds true for a primary as well as secondary immune response. Our anti-beta serum does not appear to have any anti-macrophage activity.     

Cytotoxicity in vitro of preleukaemic lymphoid cells on syngeneic monolayers of embryo or thymus cells

Lymphoid cells from preleukaemic AKR mice were cytotoxic for monolayers of syngeneic embryo and thymus target cells in tissue culture. This reactivity was detectable with cells from mice aged 3–36 weeks but was not present with cells from younger mice. Cytotoxic reactions to syngeneic embryo tissues were also seen with lymphoid cells from high leukaemia strain C3H mice carrying Moloney virus but not with lymphoid cells from normal low leukaemic strain C3H/Bi or DBA/2 mice. Lymphoid or lymphoma cells from leukaemic AKR mice showed reduced reactivity. Phytohaemagglutinin was not necessary for the reaction of preleukaemic AKR cells against AKR monolayers and cytotoxicity was inhibited by preincubation of target cells with an antiserum directed against AKR G+ cells.

The reactivity of preleukaemic AKR and C3H lymphoid cells against syngeneic monolayers may represent some type of allogeneic inhibition due to acquired antigenic differences between aggressor and target cell but the data fit best an interpretation that some lymphoid cells in preleukaemic AKR and C3H mice acquire immunological reactivity to virus-induced G+ or M+ antigens exhibited by the target cells.

     

Immunodepression in trypanosome-infected mice. VI. Comparison of immune responses of different lymphoid organs

Mitogen stimulation of cells from various lymphoid organs of C3H/He mice chronically infected with an isolate of Trypanosoma congolense was studied at different time intervals after infection, using concanavalin A (Con A) and lipopolysaccharide (LPS). At the same time, changes in the percentages of T, B and null lymphocytes in these organs were determined by immunofluorescence staining. The responses of T and B lymphocytes in the spleen were totally depressed, and the cellular composition was drastically altered by day 14 after infection. Unlike the spleen, the lymph nodes showed minor changes in their T and B lymphocyte responses and cell composition during the course of the infection, except the B cell response and composition which were altered late in the infection. The thymus and bone marrow did not show any appreciable changes in their mitogen responses and cell composition throughout the infection. The peripheral blood lymphocytes showed reduced B cell responses. Spleen cells from chronically infected mice suppressed lymphocyte stimulation induced in normal spleen and lymph node cell populations by Con A, LPS and allogeneic stimulator cells. Lymph node cells from the same group of mice did not exhibit any such suppressor activity. In the experimental system used here, the spleen is the primary site of immune depression, and other lymphoid organs such as the lymph nodes and thymus are very little affected.     

Identification by density separation of antigen-specific surface receptors on the progenitors of antibody-producing cells

Initiation of an immune response to sheep erythrocytes by the transplantation of spleen cells into irradiated recipients is thought to involve an interaction between two functionally different lymphoid cells. If such populations actually exist, it should be possible to separate them from each other and to study the properties of each type of cell. To this end, mouse spleen cells have been fractionated using equilibrium centrifugation in density gradients of Ficoll. Two functionally different populations of spleen cells were obtained. Neither population was active in initiating an immune response by itself on transplantation into irradiated mice, but mixtures of the two populations were. One population of spleen cells was also active when transplanted together with normal mouse bone marrow and the other population was active when mixed with thymus cells from normal mice. These results indicate that two different spleen cells must interact to initiate an immune response in vivo.

The specificity of the cell that synergizes with thymus cells was investigated further and shown to have antigen-specific receptors on its surface. These cells can be recognized by their ability to make rosettes with erythrocyte antigens. Thus, the `background' rosette-forming cells found in unimmunized mice are actually progenitors of antibody-producing cells.

     

Phylogeny of the lymphoid system. I. A study of the fate of circulating lymphocytes in plaice

The fate of lymphocytes collected after prolonged drainage of the neural duct of plaice (Pleuronectes platessa), labeled in vitro with [5?³H]uridine and reintroduced intravenously into the same fish was followed by means of scintillation counting and autoradiography. The characteristics of the lymphoid cell migration observed were very similar to those described in higher vertebrates. No significant numbers of labeled cells penetrated the thymus; the majority was found in the peripheral lymphoid organs, namely, the spleen and kidney. The cells lodging in the lymphoid organs had a capacity to synthesize, RNA, which the labeled cells in nonlymphoid organs lacked. The autoradiographic study revealed that the population found in the white pulp of the spleen and kidney consisted mainly of small lymphocytes, whereas the labeled large lymphocytes were present in the liver and red pulp of spleen and kidney. The relevance of the present findings to the understanding of the development of selective lymphoid cell migration is discussed.     

Studies on Mouse Auto-reactive Cells

In mice, an in vitro cell-mediated cytotoxicity is directed towards syngeneic erythrocytes used as targets. This phenomenon occurs in lymphoid organs of normal, non-immunized mice. Its intensity is greater in the thymus than in spleen or lymph nodes. The study of the nature of the cell responsible for this spontaneous syngeneic cytotoxicity ruled out the role of macrophages. The involvement of T cells in this phenomenon is assessed by its disappearance after the lymphoid celts had been treated with anti-theta serum plus complement, by its disappearance from lymphoid organs of mice previously treated by hydrocortisone, and by its decrease in the presence of synthetic thymic factor. Moreover, spontaneous syngeneic cytotoxicily is lost when lymphoid cells are depleted of autologous rosettes by centrifugation on a Ficoll-Hypaque gradient after rosette formation. Cytotoxic lymphocytes might belong to the population of auto-rosettes previously characterized as a population of immature T cells.     

Changes in lymphoid populations of ageing CBA and NZB mice

Changes in subpopulations of lymphoid cells of normal (CBA) and autoimmune (NZB) mice were studied as a function of age, by observing migration patterns of ⁵¹Cr labelled lymph node, spleen and thymus cells from donors aged 8 days to 12 months. The method permits analysis of the proportions and numbers of recirculating and non-recirculating lymphocytes in lymphoid compartments. Changes in the lymphoid populations of CBA mice were found, which could be attributed to the normal processes of maturation and senescence. In NZB mice relative and absolute decreases in the recirculating cell content of lymph node and spleen were observed which coincided with the time of development of autoimmunity. The significance of these results, in relation to altered immunocompetence with age, is discussed.     

Modification of the graft-versus-host syndrome by anti-lymphocyte serum treatment of the donor

Spleen and lymph node suspensions prepared from parental donor mice pretreated with four daily anti-lymphocyte serum (ALS) injections have been demonstrated to be incapable of inducing a graft-versus-host (GVH) syndrome when injected into F₁ hybrid recipient mice. A single ALS injection to the parental donor renders thoracic duct lymphocytes incapable of inducing a GVH response but the activity of spleen cell suspensions is retained.

It is suggested that this observation supports the view that ALS acts mainly on circulating lymphocytes.

     

Morphological and Functional Peculiarities of Lymphoid Cells in Mice of Different Strains

We revealed differences in quantitative composition and functional activity of lymphoid cells in intact mice of different strains. Cellularity and counts of lymphoid elements in hemopoietic and lymphoid organs, proliferative activity of T and B lymphocytes, and counts of CD4+ and CD8+ lymphocytes in the spleen were minimum in CC57W and Balb/c mice and maximum in CBA/CaLac and DBA/2 mice. The highest absolute content of lymphoid elements in the spleen was detected in Balb/c mice, while CC57W mice had the highest content of these elements in the thymus.     

Complete sequential regeneration of graft-vs.-host-induced severely dysplastic thymuses. Implications for the pathogenesis of chronic graft-vs.-host disease.

This study presents the sequential morphologic regeneration of graft-vs.-host (GVH)-induced dysplastic thymuses in long-term survivors of GVH reactions. GVH reactions were induced in adult C57BL/6xAF1 (B6AF1) hybrids by injecting 20 x 10(6) A strain parental lymphoid cells (PLC). Starting on day 30 after GVH induction, five to ten animals were randomly selected from a pool of GVH-reactive mice and killed at various times. Each animal was tested for thymic histology and T cell functions. Thymuses taken on day 30 after GVH induction displayed severe dysplasia as characterized by lymphocytic depletion, complete effacement of cortico-medullary demarcation, and reduction and total loss of medullary epithelial cells or both. Starting by days 60-70 after GVH induction, at least four stages of thymic regeneration were identified. Day 60-70 thymuses displayed cortical regeneration and the reappearance of cortico-medullary demarcation. The medulla of these thymuses, although containing dark individual epithelial cells and numerous lymphocytes, was devoid of pale epithelial cells (stage 1). The medulla of thymuses on day 100 after GVH induction displayed a few sparcely distributed pale epithelial cells and numerous lymphocytes as well as dark epithelial cells (stage 2). The medulla of thymuses examined 130 days after GVH induction displayed numerous pale individual epithelial cells and a few pale epithelial cell clusters. Such thymuses also showed a reduction in the number of medullary lymphocytes (stage 3). Finally, the medulla of thymuses 150-160 days after GVH induction displayed numerous pale epithelial cell clusters and Hassall's bodies. These thymuses were indistinguishable from normal adult thymuses (stage 4). All of the animals tested up to day 130 after GVH induction showed no significant T cell function. Animals displaying stage 4 of thymic regeneration showed significant proliferative responses to T cell mitogen, concanavalin A (conA), and six of ten animals also displayed a few plaque forming cells (PFC) to sheep red blood cells (SRBC) in their spleens. Furthermore, all animals (10 of 10) killed on day 180 after GVH induction displayed significant T cell functions.(ABSTRACT TRUNCATED AT 400 WORDS)