Postnatal development of mucosa-associated lymphoid tissues in chickens

Summary The postnatal development of chicken mucosa-associated lymphoid tissues of the eyes, lungs, and intestines were investigated with monoclonal antibodies specific for either all leucocytes, B lymphocytes, mononuclear phagocytes, IgM, IgG, or IgA. Attention has been paid to the relation of lymphoid infiltrates with their surrounding mucosae, the segregation into B-cell and T-cell areas, development of germinal centers, and secretory immunoglobulins. Abudant secretory IgM and IgA was detected in the epithelium of the Harderian glands in the orbits, even though they lacked large leucocyte infiltrates with germinal centers. Lymphoid tissues in the mucosae of lungs and intestines developed separate B-cell and T-cell areas. The proventriculus, Meckel's diverticulum, and Peyer's patches generally contained germinal centers from 12 weeks of age on. Because chickens as young as 2 weeks old had germinal centers in bronchus-associated lymphoid tissue and cecal tonsils, these areas were probably highly stimulated by antigens. Isotype-specific monoclonal antibodies were used to detect IgM-, IgG-, and IgA-bearing follicular cells in the same germinal center.     

Ig isotypes produced by EBV-transformed B cells as a function of age and tissue distribution

EBV can transform human B cells giving rise to lymphoblastoid cell lines that produce and secrete Ig. Herein B cells from various tissues of newborns and adults were transformed by EBV and their Ig products were analyzed with isotype-specific mAb. Although IgG- and IgA-bearing B cells were present in the newborn, EBV transformed IgM-producing cells almost exclusively in both newborn blood and breast milk. IgM-secreting cells were derived from IgM+ B cells and IgM- pre-B cells present in neonatal blood, but only from IgM+ cells in adult blood. Whereas in adults most EBV-transformed cells produced IgM, producers of IgG and of IgA were present in frequencies that varied according to the tissue source. Precursors of IgG-producing cells were relatively abundant in blood, spleen, and tonsil, and relatively infrequent in bone marrow and appendix. EBV-inducible IgA producers were relatively concentrated in the appendix and to a lesser extent in tonsils and blood. Differences in the subclass composition of EBV-transformed populations of IgG- and IgA-producers were also observed for the various adult lymphoid tissues. IgG1-producing cells predominated in most tissues, and precursors of IgG2 were largely confined to the circulation. Whereas IgA1-producing cells were predominant in all tissues, a marked enrichment in IgA2-producers was observed in the appendix. These results indicate a remarkable heterogeneity in the isotype distribution pattern of EBV-transformable B cells that is determined both by developmental age and tissue localization. We propose that EBV selectively transforms primed B cells, the isotype commitment of which varies according to tissue origin and age.     

Preliminary characterization of accessory cells in the nonadherent fraction of human blood mononuclear cells

We investigated why peripheral blood mononuclear cells rigorously depleted of adherent cells by sequential incubation on plastic and nylon wool remained fully responsive to both antigenic and mitogenic signals. Nylon wool nonadherent cells (NWNA) depleted of cells expressing HLA-DR by monoclonal antibody and complement lysis did not respond to tetanus toxoid (TT) or suboptimal concentrations of phytohemagglutinin (PHA). Addition of adherent accessory cells to these NWNA HLA-DR- cells reconstituted the response to stimuli. NWNA, fractionated by discontinuous density gradient centrifugation, contained high density cells which were unresponsive alone to optimal concentrations of both TT and PHA. All the lower density fractions contained cells which were accessory for higher density cell responses to stimuli. The lowest density fraction was approximately 30% monocytes (esterase and peroxidase positive) and less than or equal to 3% B lymphocytes (surface IgG bearing). The other low density fractions contained large granular lymphocytes but rarely monocytes and no B lymphocytes. Depletion of OKT3+, OKM1+, and Leu-11+ cells from lower density cells by monoclonal antibody and complement lysis did not abolish their accessory activity, but depletion of HLA-DR+ cells or gamma irradiation of these cells decreased their accessory activity for PHA and eradicated accessory activity for TT. Thus, the responsiveness of NWNA to soluble antigenic and mitogenic signals is due, in part, to the presence of low density cells which are radiosensitive and phenotypically HLA-DR+ OKT3-OKM1-Leu-11-. Accessory activity in NWNA seems to reside, therefore, in a cell which is not a typical monocyte, natural killer cell, nor B or T lymphocyte.     

Synthesis of immunoglobulin by rabbit lymphocytes in vitro in response to anti-immunoglobulin antiserum

Stimulation of synthesis of immunoglobulin (Ig) in vitro by Con A and anti-Ig in cultures of rabbit lymphoid cells has been analyzed qualitatively using an assay that measures the incorporation of [³H]leucine into newly synthesized proteins, followed by the specific absorption of tritiated immunoglobulin by staphylococcal protein A. Whereas Con A stimulates Ig production by spleen cells only if T lymphocytes are present, anti-immunoglobulin serum enhances Ig synthesis in the absence of T lymphocytes. In contrast, neither Con A nor anti-immunoglobulin serum stimulates peripheral blood lymphocytes to produce enhanced levels of Ig. It is concluded that both Con A and anti-immunoglobulin serum do not activate resting B cells but drive differentiation of B cells which are already synthesizing Ig. Anti-Ig acts directly whereas stimulation of B-cell Ig synthesis by Con A occurs indirectly through stimulation of T cells.     

On the mode of action of thymosin

Thymosin was administered to CBA mice which had been depleted of recirculating small lymphocytes by combining ALS and thymectomy or through lethal irradiation of thymectomised mice reconstituted with syngeneic bone marrow. The population of recirculating small lymphocytes was monitored by determining the numbers of “lymph node localising” cells in the lymphoid organs of treated animals. In no case was there any evidence that thymosin treatment accelerated the recovery of recirculating lymphocytes. Moreover, it was not possible to show that bone marrow cells incubated with thymosin acquired theta-positivity.We conclude that thymosin does not act by augmenting the production of mature recirculating small lymphocytes.     

Culture conditions affecting induction and release of lymphocyte produced proliferation inhibitory factor (PIF)

Studies on the factors affecting the production of a proliferation inhibitory factor (PIF) by human lymphocytes are presented. Maximal PIF production occurred with mitogen stimulation of blood lymphocytes cultured at 1 × 10⁶/ml. Optimal cultures contained 10% fetal calf serum, but PIF could be produced in the absence of serum, and after only a 6-hr pulse exposure to PHA. PIF production was found to correlate with lymphocyte activation in response to the mitogen PHA but was not related to lymphocyte proliferation (DNA synthesis). Inhibitory activity could be detected as early as 3 hr after mitogen addition, long before DNA synthesis occurs. The mitogens Con A and PWM initiated different intensities of DNA synthesis in these cultures, but similar quantities of PIF. Antigenic stimulation of sensitive human peripheral lymphocyte populations resulted in the release of PIF. Cells from donors that gave a strong positive skin test to tuberculin (PPD) responded in tissue culture to PPD by producing PIF, while the cells from skin test negative donors did not. A small quantity of PIF was also evident in the supernatants from cultures with no known stimulus (“unstimulated”), this was found to result from activation of the lymphocytes by nonlymphoid elements and by fetal calf serum. An investigation of the PIF-producing capabilities of other lymphoid tissues showed that lymph node cells produced this humoral factor, whereas thymus cells did not. Thymus cell supernatants, in fact, were found to contain an extremely labile cytotoxin which degraded rapidly upon storage.     

Changes in lymphocyte populations in protein—Calorie-deficient mice

Changes in lymphocyte subpopulations induced by postweaning malnutrition were studied in C57BL/6 mice kept on a protein-restricted diet (D), by weekly assessment of the “homing” properties and the response to mitogens of thymus and spleen lymphocytes during the first 2 months of diet. Cell loss in the lymphoid organs during the early phase of protein restriction was mainly due to depletion of nonrecirculating cells. This resulted in relative enrichment of medullary cells in the thymus and T₂ cells in the periphery as shown by the rise in the percentage migration of D lymphocytes to the lymph nodes as well as in their response to optimal doses of PHA and Con A and PHA:Con A response ratio. Reversion of the distribution pattern of D lymphocytes, with depressed homing to the lymph nodes and decrease in the response to mitogens, was observed concomitantly with a second phase of partial recovery in the whole-body weight and cell content of the thymus and spleen. The [³H]thymidine uptake by D spleen cells stimulated with supraoptimal doses of mitogen was significantly increased during the whole length of the experiment. The suppression of DNA synthesis induced by high doses of mitogen reappeared after short-term nutritional rehabilitation.     

In vitro response of subpopulations of human tonsil lymphocytes. I. Cellular collaboration in the proliferative response to PHA and Con A.

Human tonsil lymphocytes have been separated into three subpopulations of cells: purified B cells and two subsets of purified T cells (F1 and F2). B cells were obtained by rosetting with neuraminidase treated SRBC. F1 and F2 were separated by filtration on a nylon wool column using different speeds of elution. Purified B cells contained less than 5% T cells, the T cells preparations contained less than 5% B cells for F1 and 10 to 15% for F2, respectively. A significant contamination in cells not identified by any B or T marker was observed in purified B cells and in F1. Adherent cells enhanced the response of each lymphochte population to PHA and Con A. This explained the paradoxically low responsiveness of the purified T cells. Purified B cells did not respond to these mitogens in different culture conditions. However, a small B cell response was observed when they were cultured in the presence of mitomycin-treated T cells. Striking was the enhancing effect of B cells on the T cell response to PHA and Con A. This enhancing effect was observed even when B cells were treated with mitomycin or depleted in adherent cells. The comparison of the F1 and F2 response suggested that they contained distinct types of T cells.     

Inhibition of erythropoiesis in the spleens of irradiated mice injected with allogeneic lymphocytes. Reactivity of lymphocytes aganist non-H-2 transplantation antigens

Cell interaction requirements for generation of primary IgM, IgG and IgA responses to heterologous erythrocytes in mouse spleen cell cultures have been investigated. Interactions among antigen, macrophages, “helper” thymus-derived cells and precursors of antibody-producing cells are required and are facilitated by incubation of cultures on a rocking platform. Macrophages are required in the cultures for 48 hr for generation of optimal IgM, IgG and IgA responses. Intact erythrocyte antigen is necessary for 48 hr for development of optimal IgM responses, and for 72 hr for optimal IgG and IgA responses. Precursors of IgM antibody-producing cells appear to be “activated” by 48 hr incubation; precursors of IgG and IgA antibody-producing cells appear to be “activated” by 72 hr. These “activated” precursor cells can subsequently undergo final cycles of cell division and differentiate into mature antibody producing cells when incubated stationary in the presence of very few macrophages and in the absence of intact erythrocyte antigen.     

Transfer of immunity to Babesia microti of human origin using T lymphocytes in mice

Lymph node and spleen cells from mice infected with Babesia microti of human origin developed the ability to transfer adoptive immunity to naive mice within 25 days after infection. This protective activity was greater in cells obtained at 32 days than in cells obtained at 25 days postinfection and remained stable up to 52 days postinfection. Recipients of lymph node cells and spleen cells displayed similar peak parasitemias although 2 days after peak parasitemia, immune spleen cell recipients had significantly lower parasitemias than immune lymph node cell recipients. Strong protective activity was demonstrated when cells were transferred 1 day postinfection, while equal numbers of cells, transferred 3 days postinfection did not confer significant protection over nonimmune cells. There was also a suggestion that the number of immune spleen cells necessary for significant protection was directly related to the number of parasites inoculated. The subpopulation of lymphocytes responsible for the transfer of adoptive immunity to B. microti of human origin was then studied in BALB/c mice depleted of T lymphocytes by thymectomy and lethal irradiation. One day after infection with B. microti, T-cell-depleted mice were given complement-treated immune spleen cells, anti-θ serum-treated immune spleen cells, nonimmune spleen cells, or no cells. Similar experiments were performed comparing the effects of anti-immunoglobulin serum-treated and unfractionated immune spleen cells on B. microti parasitemia. Treatment with anti-θ serum abrogated the protective activity of immune spleen cells while anti-immunoglobulin serum treatment had no effect. These results suggest that immunologic memory of B. microti in BALB/c mice is modulated by T rather than B lymphocytes.     

Maturation of bone marrow lymphocytes. III. Genesis of Fc receptor-bearing "null" cells and B lymphocytes subtypes defined by concomitant expression of surface IgM, Fc, and complement receptors.

Lymphocyte subtypes in mouse bone marrow have been analyzed according to the combination of three surface membrane markers, IgM molecules, Fc, and complement receptors (FcR, CR), expressed simultaneously on individual cells. Marrow cell suspensions were depleted of IgM-, FcR-, and CR-bearing cells, respectively, by differential centrifugation after rosetting with appropriately sensitized erythrocytes. After rerosetting, the FcR-depleted marrow fraction showed many IgM + ve but no CR + ve small lymphocytes, the CR-depleted fraction contained both IgM + ve and FcR + ve small lymphocytes, while the IgM-depleted fraction showed many FcR + ve but few CR + ve small lymphocytes. Radioautography after [³H]thymidine labeling for 1 and 4 days in vivo demonstrated an active turnover of the various lymphocyte subtypes, particularly rapid for (IgM ? ve, FcR + ve) cells. The results demonstrate the presence of three subtypes of marrow small lymphocytes which correspond with three proposed stages in the maturation of newly formed primary B lymphocytes; (a) null cells (IgM ? ve, FcR ? ve, CR ? ve), (b) IgM + ve, FcR ? ve, CR ? ve, and (c) IgM + ve, FcR + ve, CR + ve. In addition, the turnover of a sizeable population of null small lymphocytes which bear FcR, without IgM and CR, suggests the genesis of a distinct marrow lymphocyte lineage, not previously described.     

Surface immunoglobulins on mouse myeloma cells.

Plasma cells from 22 different transplantable mouse myelomas (PCT) were tested by 14 different class-specific and type-specific anti-immunoglobulin antiserums for cytotoxicity effects using a trypan blue-exclusion method. Seven IgF,κ tumors were all sensitive to anti-IgF and anti-κ antiserums. None of the other antiserums showed a cytotoxic effect. Five IgG,κ and four IgH,κ tumors were lysed by anti-κ serums, but not by anti-IgG or anti-IgH or the others. One of two IgA,κ tumors was lysed by anti-κ, but neither was lysed by anti-IgA or the other serums.One IgM,λ tumor was lysed by anti-IgM and anti-λ. Two λ Bence Jones tumors were not lysed by anti-λ, but both of these and the IgM,λ tumor were lysed by anti-κ. One κ Bence Jones tumor was lysed only by anti-κ serum.Only the IgF tumors and an IgM tumor were lysed by the appropriate anti-heavy chain serum, and $\text{21}\text{22}$ lines had κ surface determinants.     

Inhibition of lymphocyte proliferation by monoclonal antibody directed against the T3 antigen on human T cells

Peripheral blood mononuclear cells from 40% of normal donors are mitogenically unresponsive to UCHT1, a monoclonal antibody reactive to the T3 surface molecule on human T lymphocytes. Cell preparations from non-UCHT1 responders were used to examine whether and how interaction of UCHT1 with the T3 molecule affects T-cell functionality. It was found that UCHT1 profoundly (greater than 85%) suppressed lymphocyte proliferation induced by plant mitogens (phytohemagglutinin (PHA) and concanavalin A (Con A], recall antigen (candidin), and allogeneic non-T cells. The antibody abrogated both the production of interleukin 2 (IL-2) by and the expression of IL-2-specific receptors on T lymphocytes stimulated by PHA or allogeneic non-T cells. UCHT1 was maximally suppressive when added to cells within 2 hr (PHA stimulation) or 1 day (allogeneic non-T cell activation) after the initiation of the culture period. The inhibiting activity of UCHT1 could be related to its ability to modulate T3 molecules from the T-cell surface: both actions displayed the same antibody concentration dependence and had a comparable time dependence. Moreover, after modulation, unresponsive lymphocytes regained responsiveness to PHA in parallel with reexpression of surface T3 molecules. These findings are consistent with the idea that the human T3 molecule functions as an essential signal transducer during the early phases of T-cell activation.     

Presence of a reversible inhibitor for human lymphoid cell DNA synthesisin the extracts of human peripheral lymphocytes.

An endogenous inhibitor of human lymphocyte DNA synthesis contained in extracts of purified human peripheral lymphocytes is described. It was found that the peripheral lymphocyte extract inhibits the DNA synthesis of phytohemagglutinin (PHA) stimulated human peripheral lymphocytes, lymphocytes in mixed lymphocyte culture, and human lymphoid cells in a long-term culture (PGLC-33H). This extract did not inhibit the DNA synthesis of nonlymphoid cells including HeLa and human embryonic lung. The effects of the inhibitor were reversible and noncytotoxic. Initial characterization showed the inhibitor to be thermolabile, DNase resistant, trypsin sensitive, and stable in a pH range 5.4–8.4. It appears that the inhibitor contained in the purified human peripheral lymphocyte extract is similar to a previously described inhibitor extracted from a human lymphoid cell line (PGLC-33H). Quantitation of the inhibitor in various lymphoid cell populations showed the amount of inhibitor per cell to be higher in resting peripheral lymphocytes than in PHA stimulated peripheral lymphocytes or human lymphoid cells in long-term culture (PGLC-33H). This data suggest that the inhibitor described may play a regulatory role in lymphocyte metabolism.     

Phylogeny of lymphocyte heterogeneity. II. Differential effects of temperature on fish T-like and B-like cells.

Lymphocytes from the bluegill, a freshwater fish, were observed to undergo in vitro mitogenic responses to a variety of “classical” mitogens. Using cell fractionation approaches based upon surface markers and in vitro mitogenesis, bluegill lymphocytes could be divided into two populations. One population responded to PHA and Con A but not to LPS, contained surface antigens in common with bluegill brain, and did not form spontaneous rosettes with rabbit erythrocytes. The other population responded to LPS but not to PHA or Con A, did not appear to contain surface antigens in common with brain, and did form rosettes with rabbit erythrocytes. The former population responded to mitogenic stimulation very well at 32 °C, whereas the latter population responded better at 22 than at 32 °C. The pattern of mitogenic responses and brain antigen distribution coupled with the observation that mixed lymphocyte responses were obtained at 32 but not at 22 °C makes it likely that the 32 °C responsive population represents the fish equivalent of T cells. The other population may be B cells. These data suggest that the immunosuppressive effects of low temperatures on cold-blooded animals may be effects on the generation of functional T cells and not on B cells.     

Tissue distribution of immunoglobulin-secreting cells in normal and IgA-deficient chickens.

A reverse hemolytic plaque assay was employed to enumerate lymphoid cells actively secreting either immunoglobulin (Ig)G, IgA, or IgM in the small intestine, lungs, and lymphoid organs of normal and IgA-deficient chickens. In normal birds, intestinal lamina propria lymphocytes were proportionately richest in cells secreting IgG and IgA whereas the bone marrow was richest in IgM-secreting cells. The highest ratio of IgA to IgG secreting cells was also found in the lamina propria lymphocytes of the intestine (0.9), followed by the IgA to IgG ratios in the intestinal epithelium (0.31), and the lungs (0.19). The IgA to IgG ratios in the bone marrow (0.08) and the spleen (0.02) were considerably lower. Thus, both the intestine and the lungs were relatively enriched in cells actively secreting IgA. These IgA-secreting cells are the likely source of the IgA found in such quantities in intestinal and respiratory secretions. The tissue distribution of Ig-secreting cells was also studied in two generations of birds with experimentally induced IgA deficiency. There was a striking diminution of IgA-secreting cells in all tissues, including the intestine and lungs, whereas cells secreting IgG and IgM were normal or increased. The lack of IgA-secreting cells in these birds represents the effects of donor suppressor T cells having specificity for IgA.     

Separation of human lymphocyte subpopulations by density gradient electrophoresis. I. Different mobilities of T (T mu, T alpha) and B lymphocytes from human tonsils

T and B lymphocytes from human tonsils were separated by density gradient electrophoresis on the basis of their surface charge. The high-mobility cell fractions were found to be highly enriched in T lymphocytes with only very small proportions of B cells. In contrast, the low-mobility fractions were predominantly B lymphocytes, and had only 10 to 30% contamination of T cells. The intermediate-mobility fractions contained both T and B lymphocytes in approximately equal proportions. IgM-bearing lymphocytes, as well as cells with receptors for mouse erythrocytes, the Fc portion of IgG, and complement were found in the intermediate- and low-mobility fractions. T lymphocytes, prepared by E rosetting, were also electrophoresed by this method and found to be of higher mobility as compared with peripheral blood T lymphocytes. T cells with Fc receptors for IgM (Tμ) or IgA (Tα) were found to be considerably heterodisperse with regard to surface charge and were present in all fractions. The separated cell fractions were treated in vitro with various concentrations of concanavalin A and thereafter examined for Tμ, Tγ, and Tα phenotypes. Low concentrations of Con A (2.5 μg/ml) had no effect on cell surface phenotypes. However, higher concentrations of Con A (20μg/ml) significantly reduced the numbers of T cells having IgM receptors (Tμ), but failed to alter the expression of the Tγ phenotype. The latter finding contrasts to that observed with T cells from the peripheral blood where high concentrations of Con A increase the proportions of the Tγ cells. This study demonstrates that density gradient electrophoresis can be used for the separation and study of lymphocyte subpopulations from human tonsils.     

Regulation of IgA secretion by T cell clones derived from the human gastrointestinal tract

The role of T cells in Ig isotype regulation is still unclear. To address this question, we generated mitogen-stimulated T cell clones from normal human lymphoid follicles of the gut-associated lymphoid tissue (appendix). Both the T cell clones and clonal supernatants provided preferential help for IgA secretion by PWM-stimulated B cells. Many of these CD3+, CD4+, 4B4+, DR+ helper clones co-expressed Fc-gamma and Fc-alpha R, but there was poor correlation between the expression of Fc-alpha R and IgA help (p = 0.31). Most of the T cell clones helped both IgM+A- and IgM-A+ B cell populations to secrete IgA, suggesting that they mediate switch of isotype-uncommitted B cells as well as post-switch expansion of IgA-committed B cells; however, some of the T cell clones helped IgM+A- B cell populations much more than IgM-A+ B cell populations, suggesting that, in this case, the regulatory effect is predominantly at the level of B cell switch. In all, these results show that the mucosal immune system contains individual T cells which are capable of positively regulating IgA-specific isotype differentiation at two levels of B cell development, thus allowing for efficient generation of IgA-secreting B cells.     

Electrophoretic separation of rat spleen and thymus leukocytes and their reactions to mitogens in vitro]

Electrophoretic mobility (EPM) of lymphocytes from the thymus and spleen of August and Wistar rats as well as capacity of lymphocytes with different surface hemagglutinin (PHA) and concanavalin A (Con A) were studied by the method of free flow electrophoresis. Lymphocytes of the rat spleen were shown, depending on the surface charge, to divide into two groups during cultivation: cells with high and low electrophoretic mobility. At separation the lymphocytes consisted of 8--10 fractions with different EPM. There was a relationship between the surface charge of the lymphocytes and their stimulation rate by mitogens. Increased thymidine-3H uptake was recorded at mitogenic exposure of lymphocytes from the spleen with high EPM. Low mobile lymphoid elements of the spleen did not respond to mitogenic stimulation. A subpopulation of thymocytes with low EPM was resistant to Con A stimulation. The thymocytes of rats did not virtually respond to PHA irrespective of EPM.     

In vitro study of IgM polymerization.

The polymerization of 7S IgMs from normal rabbit lymphoid cells, stimulated either with antigen or with mitogen (Con A), has been studied. The process was analyzed by characterizing the various molecular forms by sucrose gradient sedimentation and susceptibility to anti-μ serum and 2-mercaptoethanol. It has been shown that native J chain and an enzyme are both required for the proper assembly of IgM pentamer. The enzyme preparation (PMF) is active only if it is extracted from spleen cells stimulated to IgM production. When the extract is prepared from nonstimulated lymphoid cells, or from liver cells, incubation of IgMs with PMF does not lead to the formation of 19S IgM, but to molecules of intermediate size and to various aggregates. It is shown that antibody activity of IgMs and of these heterogeneous polymers are not susceptible to treatment with 2-mercaptoethanol. In contrast, antibody activity of the pentameric IgM is completely inhibited by 2-mercaptoethanol. A PMF inhibitory substance was present in the postmicrosomal supernatant. When added in the incubation medium, this substance prevented the proper polymerization. Its eventual role in IgM biosynthesis in nonstimulated, and specifically stimulated cells is discussed compared with mitogen stimulated cells, and tumor lymphoid cells.