The proportions of B and T lymphocytes in lymphomas, peripheral nerves and lymphoid organs in Marek's disease

The proportions of cells bearing B cell, T cell and immunoglobulin (Ig), cell membrane markers have been studied in chickens with Marek's disease (MD). The proportions of B, T and Ig-bearing cells were similar in lymphomas arising in various organs and in infiltrated nerves, irrespective of type of nerve lesion. T cells provided the major component, and B and Ig cells the minor components, of the lymphoid cell populations. Compared with the corresponding organ from uninfected control chickens, bursa and spleen from MD-affected birds showed increases in the proportions of T cells and decreases of B and Ig cells. In the bursa, and probably in the spleen, the increase in the proportion of T cells was caused by lymphomatous infiltration. In the thymus of MD-affected chickens the proportion of T cells was decreased, and of B cells increased.     

Solubilization and partial purification of lymphocyte specific antigens in the chicken

Four different lymphocyte antigens were solubilized from chickens homozygous at the B locus. The antigens were identified by immunoelectrophoresis with rabbit anti-lymphocyte sera. 1. A bursa-specific cell surface antigen was extracted with 3 M potassium chloride and partially purified. The antigen was excluded from Sephadex G-200 gels, was heat labile and was a potent immunogen. The antigen could be detected on all bursa cells by immunofluorescence but not on thymus, spleen or blood lymphocytes. The findings do not exclude, however, the possibility that a small number of lymphocytes (e.g. plasma cells) possess the antigen. 2. A thymus-specific antigen was obtained by papain treatment of thymus cells. It migrated cathodically in immunoelectrophoresis. 3. A surface antigen which was specific for lymphocytes from both central lymphoid organs (thymus and bursa) was solubilized by pestle homogenization. 4. A fourth lymphocyte surface antigen was present on lymphocytes from blood, spleen, bursa and thymus. It was best solubilized by 3 M potassium chloride extraction and did not migrate under the conditions of immunoelectrophoresis. Studies did not reveal a stable antigenic marker with specificity for thymus or bursa cells and their progeny in the peripheral lymphocyte pool.     

Comparative natural killer cell activities of thymic, bursal, splenic and intestinal intraepithelial lymphocytes of chickens

The natural killer (NK) cell activities of spleen, thymus, bursa, peripheral blood and gut intraepithelial lymphocytes (IEL) from FP and SC chickens were investigated in 4-hr and 16-hr 51Cr release assays. Target cells were 4 different tumor cell lines derived from either an avian leukosis tumor transplant (LSCC-RP9, LSCC-RP12) or from Marek's disease lymphomas (MDCC-MSB-1, MCDD-CU36). Great variability in cytotoxic potential was observed among NK cells of different lymphoid organs. NK cell cytotoxicity varied depending upon the type of effector cells, type of target cells, the ratio of effector to target cells, and the age and genetic background of chickens. Substantial levels of NK cell activity were detected in spleen and gut IEL of SC chickens in a 4-hr assay. In contrast, the NK cytotoxicity in gut IEL of FP chickens was not detectable until 16 hr after incubation. The ranges of target cell specificity demonstrated by IEL, spleen, thymus and bursa NK cells were similar to one another and, in general, the level of cytotoxicity increased with incubation time. Thymus and bursa NK cell activity of both SC and FP chickens was not detectable in a 4-hr assay but substantial NK cell activity was demonstrated in a 16-hr assay. The results of the present study demonstrate that various lymphoid organs of chickens, such as spleen, thymus, bursa, and gut intraepithelium, contain subpopulations of cells that can mediate spontaneous cytotoxicity.     

Cellular heterogeneity in the thymus. I. Electrophoretic analysis of lymphoid cell populations from chickens

Micro-cell-electrophoresis has been applied to demonstrate differences and heterogeneities in various lymphocyte populations. The lymphocytes from the blood, spleen, bursa and thymus of young chickens differ significantly in their electrophoretic mobility (EM) pattern. The blood and spleen lymphocytes appear to be homogeneous by this criteria; bursa cells show slight heterogeneity, and thymus lymphocytes contain two clearly distinct subpopulations. One of the two thymocyte subpopulations is absent in neonatally bursectomized and irradiated animals. This bursa-dependent cellular component in the thymus has about the same EM as bursa cells themselves, which may suggest that these cells are, as such, derived from the bursa. The possible role of the bursa-dependent subpopulation is discussed. Within the first months of life, there is a dramatic shift from the predominance of the electrophoretically slower population to its virtually complete disappearence and the subsequent dominance of the other cellular component. Bursectomy plus irradiation provided pure preparations of one of the thymus subpopulations. The other subpopulation could be highly enriched by differential centrifugation in chicken plasma. Both subpopulations are thus separately available for direct immunological tests. The reduction of the EM by neuraminidase show that sialic acid is a major charged component in the cell surface of all lymphocyte preparations tested. The data indicate also that the different EM of the two thymocyte subpopulations is not due to different amounts of neuraminidase labile groups on the cell surface. The high EM of blood lymphocytes, on the other hand, is probably to be explained by a high content of neuraminidase-labile groups, as well as some ribonuclease sensitive material. A substantial reduction of the EM by ribonuclease was observed on blood lymphocytes, but not on lymphocytes from other organs.     

Effects of fowl adenovirus infection on the immune system of chickens.

A virulent strain of serotype 8 fowl adenovirus (FAV) was isolated from an outbreak of inclusion body hepatitis (IBH) in broiler flocks. Post-mortem changes included characteristic liver lesions with intranuclear inclusion bodies in the hepatocytes and severe lymphocytic depletion in the bursa, thymus and spleen. The packed cell volume was reduced by 50 per cent or more and varying amounts of cell depletion were observed in the bone marrow. Typical IBH was reproduced in specific pathogen-free chickens inoculated orally with the FAV isolated from the natural infection. There was severe depletion of lymphocytes in the bursa, thymus and spleen of the experimentally infected birds and FAV antigens were detected by ELISA and immunocytochemical staining in various lymphoid tissues. Humoral antibody responses against sheep red blood cells, detected by the haemagglutination test, were decreased in the chickens infected with FAV. These findings suggest that the damage caused by replication of this virulent strain of FAV in lymphoid tissues compromises the immunological capabilities of infected chickens.     

Surface immunoglobulins on the lymphocytes of the skate Raja naevus

Living lymphocytes of the Elasmobranch fish, Raja naevus, have been examined for surface immunoglobulin (Ig) by treatment with a fluorescent anti-Ig system. Large numbers of Ig-positive cells (60-80%) were found in peripheral blood, spleen and thymus. Following modulation of the surface Ig with anti-Ig, resynthesis occurred, showing that the surface Ig is a product of the individual lymphocytes rather than material passively absorbed from the serum. Formation of caps was independent of temperature, occurring as readily at 4 degrees C as at 20 degrees C, a finding which presumably reflects the environmental conditions normally experienced by the skate. The presence of Ig-bearing lymphocytes in the adult skate thymus suggests a similarity to the amphibian larval thymus, which may be a primary lymphoid organ for the production of both T and B lymphocyte analogues.     

Receptors for activated C3 on thymus-dependent (T) lymphocytes of normal guinea-pigs.

In a survey of lymphocyte subpopulations in normal guinea-pig blood, lymph node, spleen, thymus and peritoneal cavity, a considerable overlap was observed between the percentages of C3-receptor bearing lymphocytes (CRL) and of thymus-dependent (T) cells in lymph nodes. Simultaneous rosette-formation reactions with sheep erythrocytes carrying rabbit complement (EAC) and papain-treated rabbit erythrocytes (a T-cell marker) revealed that 20--50% of the lymph node CRL were T lymphocytes. These experiments and others on cell suspensions depleted of Ig-bearing (B) lymphocytes showed that between 8 and 36% of lymph node T cells have complement receptors. The frequency of T-CRL in other lymphoid tissues was lower, representing between 0 and 8% of the T-cell population. The reaction of T-CRL and EAC was not inhibited by EDTA which is known to inhibit the C3 receptor activity on macrophages.     

Evaluation of the possible role of B cell receptors in the tendency of B cells to migrate into follicles in mice and chickens.

Chicken spleen and bursa cells were examined for the percentage of Fc receptor-bearing cells. Rosette formation was done with chicken 7S antibody-sensitized sheep erythrocytes and was inhibited by heat-aggregated chicken Ig. In the spleen, the percentage was found to increase with age to approximately 26% at 7 to 12 weeks. In contrast, only 3 to 5% of bursa cellss at this age demonstrated Fc receptors. Sleens from bursectomized chickens had 7--10% Fc receptor-bearing cells. In an attempt to determine a possible role of the C3 receptor on migration patterns, the effect of cobra venom factor (CVF) on the localization of transferred lymphoid cells was examined. Pretreatment of recipients with enough CVF to lower mean C3 levels to 11% of controls failed to affect follicular B cell localization in mice at either 24 or 48 h after transfer. Localization of thymus or bursa cells in chickens was similarly unaffected by CVF pretreatment. The possible roles of Fc and C3 receptors on migration of B lymphocytes into follicles and germinal centers were discussed.     

Surface immunoglobulin on granular and agranular leukocytes in the thymus and spleen of the snapping turtle, Chelydra serpentina

Spleen and thymus suspensions from the turtle, Chelydra serpentina were examined by indirect immunofluorescence and found to contain both agranulocytes and granulocytes positive for surface Ig. Among the splenic agranulocytes , 50% of the lymphocyte and monocyte population was positive for surface Ig. In the thymus only 7% of the thymocytes were positive. Ninety-two percent of the granulocytes, composed primarily of basophils and eosinophils, were positive for surface Ig in both the thymus and spleen. The presence of surface immunoglobulins on turtle splenic leukocytes was confirmed by sheep red blood cell (SRBC) immunization studies. Basophils, lymphocytes and monocytes from immunized turtles formed SRBC rosettes, while eosinophils from immunized turtles were found to specifically phagocytose SRBC. Splenic leukocytes from control turtles did not phagocytose or rosette with SRBC. This study demonstrates that indirect immunofluorescent techniques can be used to identify surface immunoglobulin on turtle granulocytes as well as agranulocytes .     

Antigenic surface determinants of chicken lymphoid cells. I. Serologic properties of anti-bursa and anti-thymus sera

The serologic properties of turkey antisera to chicken bursa (ABS) and thymus (ATS) cells were assayed by cytolysis-in-agar, conventional cytotoxicity tests and indirect immunofluorescence. Using proper absorptions the following antigenic surface determinants were detected on bursal and thymic lymphoid cells: (a) common lymphocyte antigens present on both kinds of cells; (b) thymus-specific antigens; (c) bursa-specific antigens; (d) in addition to the latter, bursa cells displayed immunoglobulin surface determinants. Even before absorption the anti-thymus and anti-bursa sera gave higher titres of reactions with homologous target cell preparations. In complement dependent cytotoxicity tests ABS and ATS reacted specifically with bursa and thymus cells respectively. In the spleen of 2- and 3-week-old chickens 20–30% of lymphoid cells were killed by ABS and 40–50% by ATS. The advantage of avian antilymphocyte sera for in vivo studies in chickens are emphasized.     

Influence of specificity of reagents and of behaviour of cell surface Ig on the detection and enumeration of immunoglobulin-bearing lymphocytes in humans.

Some parameters likely to influence detection and classification of human B-lymphocytes using anti-immunoglobulin (Ig) sera have been investigated. Of 20 separate mono- and polyspecific native or conjugated anti-Ig sera analysed by a passive haemagglutination technique, 13 exhibited non-specific reactivity. This technique showed no consistent correlation between the titre of individual sera against Fab2 and whole IgG respectively. The indirect immunofluorescence (IF) method applied to detect surface Ig on blood lymphocytes seemed to detect Fc-bearing rather than Ig-bearing cells. The direct method generally yielded fewer reacting cells (5%) than the indirect (10-25%), suggesting that Fc-bearing cells are more numerous than Ig-bearing cells. The Ig-bearing blood lymphocytes seemed to belong preferentially to the IgM class. Passively adsorbed Ig did not appear to contribute significantly to the number of Ig-bearing cells detected. Anti-Ig sera induced redistribution and some endocytosis of surface Ig but this did not markedly affect detection of Ig-bearing cells.     

Cellular heterogeneity in the thymus. II. Distribution patterns of different lymphocyte populations in chickens

Lymphocyte preparations from chicken blood, spleen, bursa, as well as fractionated thymus cell preparations, were labeled in vitro with ⁵¹Cr and reinjected into various groups of recipients. The relative distribution patterns of labeled cells in numerous recipient organs remained practically unchanged after different time intervals, but were significantly different for the various cell preparations. The distribution patterns provided additional evidence for the existence of different lymphocyte subpopulations in the thymus of young chickens, one of which is bursa-dependent. Thymus cell preparations fractionated by differential centrifugation showed significant differences in their accumulation in the spleen and liver. The analogous lymphocyte preparations from neonatally bursectomized birds showed no such differences. Cells from 6-month old donors gave results similar to those obtained with cells from young bursectomized birds, suggesting that the bursa-dependent subpopulation in the thymus disappears after involution of the bursa. An unexpected finding was the rather large accumulation of blood lymphocytes in the thymus of the recipients. This observation stands in contrast to the concept of a blood-thymus barrier.     

Pathology of lymphoid organs in chickens fed a diet deficient in zinc.

An experiment was conducted, including flow cytometry, to study the pathology of the lymphoid organs and peripheral blood T lymphocytes in zinc (Zn)-deficient chickens. One hundred 1-day-old broiler chickens were randomly divided into two groups and fed on diets with 100 mg/kg Zn (controls) or Zn-deficient diets (Zn, 23.63 mg/kg) for 7 weeks. The weight and growth index of the bursa of Fabricius, thymus and spleen were significantly reduced (P<0.05 or P<0.01) in Zn-deficient birds when compared with those of control broilers. The G0/G1 phase of the cell cycle of the bursa, thymus and spleen was much higher (P<0.01), and the S, G2+M phases and proliferating index lower (P<0.05 or P<0.01) in Zn-deficient broilers than in the controls. The acid alpha-naphthyl acetate esterase-positive ratio of the peripheral blood T lymphocytes and the CD4 and CD8 numbers were markedly reduced (P<0.05 or P<0.01), and the CD4/CD8 ratio increased. Histopathologically, lymphocytes of lymphoid organs were depleted and the reticular cells of the thymus were also degenerate or necrotic in the Zn-deficient birds. The results demonstrate that Zn deficiency seriously inhibited the development of lymphoid organs, impaired the progression of lymphocytes from the G0/G1 phase to the S phase, and caused pathological injury in the lymphoid organs. The results also showed that the effect of Zn deficiency on the primary lymphoid organs occurred earlier than on the secondary lymphoid organs. The effect of Zn deficiency was greatest on the bursa of Fabricius, followed by the thymus, and then the spleen. Potential mechanisms underlying the observations are discussed.     

Ontogeny of lymphoid cell surface determinants in the chicken.

Five antigenic lymphoid cell surface determinants (LCSD) were detected in hatched chickens using specific antisera. These LCSD were: thymus-specific surface determinants, bursa-specific non-immunoglobulin determinants, IgM-specific determants, IgG-specific determinants, and IgA-specific determinants (ASD). Viable cell suspension of embryonic yolk sac, bursa, thymus and spleen were tested by means of indirect or direct immunofluorescent staining procedures for the presence and frequency of LCSD during maturation. Experiments performed with liver cells. brain cells and red blood cells of embryos confirmed the specificities of the antisera used for determinants present on cells of lymphoid tissues. The results showed LCSD to occur on yolk sac cells on the 5th to 7th embryonic day (ED). This suggests the presence of a stem cell pre-committed for the lymphoid cell line already in the yolk sac. Furthermore, findings are reported indicating the presence of distinct lympoid stem cell populations or maturation stages in the yolk sac, which may be responsible for either populating the thymus or the bursa. The finding of ASD-bearing cells early in ontogenesis of the lymphoid system suggests the presence of two specificities in anti-chicken IgA sera, one of which may be directed against an antigenic site on a rudimentary immunoglobulin molecule, which becomes lost or hidden in later maturation. Studies on the bursa and the thymus show that covering, hiding, or loss of antigenic determinants plays an important role in lymphoid cell differentiation. Furthermore, the spleen is reached by B-determined stem cells as early as the bursa, but these stem cells seem not to proliferate in the former to any considerable extent until hatching. Finally, the sequence of the appearance of immunoglobulin classes as proposed by other authors is confirmed with reservaitons concerning IgA, and it is suggested that immunoglobulins are detectable earlier on cell surfaces than intracytoplasmatically.     

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.

     

Role of the spleen in the pathogenesis of Marek's disease

The importance of the spleen for the pathogenesis of Marek's disease (MD) was investigated. The spleen was not required for infection of other organs or for the development of latently infected peripheral blood lymphocytes (PBL). Splenectomised (Sx) chickens had infected lymphocytes in the thymus, bursa and PBL by 6 or 7 days after intratracheal exposure to MDV. However, splenectomy did delay the spread of infection since intact birds had positive cells in these tissues 2 to 3 days earlier. Splenectomy reduced the number of visceral tumours in JM-10- but not in GA-5-infected chickens. Splenectomy did not influence the degree of protection by vaccination with SB-1.     

Immunological capacity of the chicken embryo. I. Relationship between the maturation of lymphoid tissues and the occurrence of cell-mediated immunity in the developing chicken embryo.

In an investigation of the ontogeny of lymphoid tissue in chick embryos to relate maturation of lymphocytes with immunological competence, the numbers and sizes of lymphocytes were determined in the thymus, bursa of Fabricius, spleen, femoral marrow and peripheral blood of embryos from the 12th to 21st day of incubation, and in 6-day-old chicks. Results showed the thymus to be the first fully developed and most active lymphocytopoietic organ, followed by the bursa. The bone marrow was not lymphocytopoietic; the spleen and bone marrow were mainly granulocytopoietic and erythropoietic; some morphological differences between thymic and bursal lymphocytes were shown by light microscopy. It appears that in embryos and young chicks the lymphocytes are derived from the thymus and bursa, but not the bone marrow. In tests of immunological competency, cells of the thymus, bursa, spleen, bone marrow and peripheral blood from 12--21-day-old embryos and 6-day-old chicks were transferred to chorioallantoic membranes of 12-day-old recipient embryos. There were distinct differences between the ability of various lymphoid tissues to induce formation of chorioallantoic pocks or splenic enlargement. The thymus, spleen and peripheral blood elicited both lymphocytic pocks and splenomegaly, the bursa elicited splenomegaly only, and the bone marrow was ineffective. The bone marrow, however, induced formation of nonlymphocytic pocks. It is concluded that the immunological activity of the chicken embryo is primarily effected by the thymus and bursa and that cell-mediated immunity appears in the 2nd week of incubation.     

The formation of immunoglobulins by human tissues in vitro: III. Spleen, lymph nodes, bone marrow and thymus

The site of immunoglobulin formation in human tissues was studied with three techniques: incubation of tissue fragments with radioactive amino acids followed by autoradiographic analysis of the synthesized immunoglobulins, immunofluorescent staining of tissue sections and cell suspensions, and morphological examination.

These studies showed that the spleen, lymph nodes and bone marrow synthesize IgG, IgA and IgM. In agammaglobulinaemia, one bone marrow sample showed no immunoglobulin synthesis, the other sample synthesized a trace of IgG. Immunofluorescent staining has demonstrated that in the spleen and lymph nodes plasma cells and large and medium-sized lymphocytes were positive for IgG, IgA or IgM; small lymphocytes were only positive for IgM. In bone-marrow samples, however, only plasma cells were positive for immunoglobulins. It is discussed whether in the bone marrow the cells that synthesize immunoglobulins do not originate in this organ, but derive from other lymphopoietic organs.

The normal thymus showed a different pattern because it synthesized only IgG and IgA. The plasma cells and medium-sized lymphocytes, which synthesize immunoglobulins, were localized predominantly in the interstitial connective tissue and ocasionally in the medulla, both near blood vessels. In all likelihood these cells did not originate in the thymus, but were trapped in this organ from the circulation. In autoimmune diseases, however, the thymus showed IgM-positive germinal centres and plasma cells and synthesized IgG, IgA and also IgM.

     

Emigration of B cells from chicken bursa of Fabricius

The extent of emigration of cells from the bursa of Fabricius to the periphery was estimated. Per anum application of fluorescein isothiocyanate to label bursal cells in situ was used. Migrant cells can be visualized on frozen sections or cell suspensions of peripheral organs by their fluorescence. The data show that at 2-3 weeks after hatching about 5% of bursal cells leave the bursa per day. Since the bursal cells divide rapidly, this indicates that the vast majority (95%) of bursal cells die in situ. Cells that leave the bursa are surface IgM positive and go first to peripheral blood and later into B cell areas of spleen, thymus and cecal tonsils. The results are also discussed on the basis of their implication for the generation of antibody diversity in the chicken bursa.     

Identification of a subpopulation of chicken B lymphocytes by the lectin from Lotus tetragonolobus

Lectin-reactive chicken lymphoid cells were detected by agglutination. The lectin from Lotus tetragonolobus agglutinated cells from bursa and spleen and did not agglutinate cells from thymus or peripheral blood of 28-day-old chickens. The percentages of Lotus tetragonolobus reactive cells were also measured by assays of lectin-induced rosette formation, binding of lectin-labelled latex beads, and binding of rhodamine-labelled lectin. The distribution of lectin reactive cells varied with the age of the chicken. The lectin appears to identify a unique subpopulation of chicken B lymphocytes.