Immunofluorescence studies on the expression of VH a allotypes by pre-B and B cells of homozygous and heterozygous rabbits

Fluorochrome-conjugated antibodies specific for C mu determinants and VH a allotypes were used to examine pre-B cells and B lymphocytes for expression of these markers. The majority of mu+ bone marrow pre-B cells were shown to express a allotypic determinants in conjunction with C mu. Both pre-B and B cells from a2 a3 heterozygous rabbits showed allelic exclusion of these allotypes. Pre-B cells expressing the a2 or a3 specificities appeared to be generated in approximately equal numbers in heterozygotes, while B lymphocytes expressing a3 appeared to undergo preferential clonal expansion very early in development. It was also observed that rabbit bone marrow and blood contained a population of myeloid cells which, in a2 a3 heterozygotes, stained for both a2 and a3 determinants. The frequencies of this cell type, which exhibited bright immunofluorescence staining for a allotypes, could not be reduced by prolonged incubation at 37 degrees C but were readily reduced after cell suspensions were treated with low pH buffer. It is concluded that these myeloid cells bear high avidity Fc receptors for serum immunoglobulin and may be the culprits in studies which have found production of two a or b allotypes by individual B lymphocytes.     

Quantitative distribution of group A allotypes in normal heterozygous rabbits

Normal serum samples from rabbits heterozygous for the group a allotypes were studied using a sensitive radioimmunoassay inhibition technique to determine concentrations of immunoglobulin molecules bearing a1, a2, or a3 epitopes. Three groups of sera representing each of the possible group a heterozygous combinations were studied. Results showed quantitative differences in allotype expression manifested by an ordered preponderance of a1 > a3 > a2. These data provide evidence to support theories that regulatory control mechanisms mediate allotype expression.     

Functional diploidy for the secretion of the a allotypes by the lymphoid cell of heterozygous rabbits

Can a given heterozygous lymphoid cell, for the ‘a’ system of allotypes in the rabbit, secrete immunoglobulin molecules of the two types (functional diploidy) with the same antibody specificity? This problem was investigated by two methods (applied to immunized lymph node or spleen cells): enhancement of plaques of hemolysis by separate action of two anti-allotype antibodies vs the effect of a mixture of antisera, or by micromanipulation.

Both methods lead to the same results: many cells manifest functional diploidy.

In addition, it was found that only two populations of cells existed: cells secreting only al and cells secreting a1 and a2 or a1 and a3.     

Antigenic determinants on bovine encephalitogenic protein. Localization of regions that induce transformation of lymph node cells from immunized rabbits

With the aid of a lymph node cell transformation test and several fragments of the bovine encephalitogenic protein, attempts have been made to examine more closely various parts of the protein that stimulate DNA-synthesis of lymph node cells from immunized rabbits. Evidence was obtained for the presence of at least two strongly stimulating regions within residues 44–170: one resides in peptide 44–89 and the other in peptide 93–170. When parts of these regions and other oligopeptide derivatives of region 44–170 were tested, only a slight or no stimulating effect was observed, and a closer localization of all reactive sites – corresponding to that made in guinea pigs with the macrophage migration inhibition technique – could not be made unequivocally. However, peptides 44–68 and 154–170 were shown to have transformation-inducing effects. Further evidence for the nonreactivity of the tryptophan region was obtained in accordance with previous findings. For at least one region of the encephalitogenic protein – the tryptophan region – there is thus no correlation between disease-inducing activity and ability to stimulate lymph node cells from immunized rabbits to transform in vitro.     

Suppression of the paternal IgA-f and IgA-g allotypes in heterozygous rabbits suppressed for the paternal IgM allotype

The injection of neonatal rabbits of genotype a¹n^{8 1}g^{7 4}/a²n^{8 2}f⁷¹g⁷⁵with anti-n81 antiserum suppressess the expression of the paternal f73 and g74 IgA allotypes; the expression of the maternal 171 IgA allotype is enhanced but the expression of the g75 allotype is not significantly enhanced. The concentrations of these allotypes in serum correlated with the immunofluorescent analyses of cellular ratios in gut sections. Allotype suppression of IgA with an anti-IgM reagent supports the concept that the IgM bearing cells are the precursors of the IgA secreting cells.     

Expression of kappa chain allotypes at the cell surface and in serum immunoglobulins of normal and allotype-suppressed heterozygous rabbits

The kappa light chain allotypes b4 and b5 were measured in the serum and on the surfaces of peripheral blood lymphocytes of normal and allotype-suppressed heterozygous rabbits. Surface immunoglobulins (sIg) were detected by fluorescence microscopy and additional quantitative data were obtained by flow microfluorometry. Although a few b5-suppressed animals had no b5 in serum or on cell surfaces for years, most b5-suppressed and all b4-suppressed animals studied had some cells with sIg of the suppressed type by 1 year of age. In suppressed animals the level of serum Ig remained depressed throughout life but cells with sIg appeared in disproportionately large numbers. The effect was particularly striking in those animals suppressed for the b4 type.     

Localization of antigen-specific lymphocytes following lymph node challenge.

The effect of subcutaneous injections of Brucella abortus strain 19 antigen on the specific localization of autologous lymphocytes in the regional nodes of calves was analysed by fluorescent labelling and flow cytometry. Both in vitro and in vivo FITC labelling of lymphocytes indicated the preferential migration of lymphocytes from a previously challenged lymph node to a recently challenged lymph node. However, lymphocytes from a lymph node challenged with B. abortus failed to localize preferentially in a lymph node challenged with a control antigen, Listeria monocytogenes. Lymph node cells, enriched for T lymphocytes and isolated from primary stimulated or secondary challenged B. abortus lymph nodes, could proliferate when cultured with autologous antigen-pulsed macrophages. The kinetics of [3H]thymidine incorporation in lymphocytes from secondarily challenged lymph nodes occurred earlier and to a greater extent when compared with lymphocytes from primary challenged lymph nodes. Our data show that the accumulation of B. abortus-specific lymphocytes in secondarily challenged lymph nodes is increased by the presence of the specific antigen.     

Benign mesothelial cells in lymph node

This commentary summarizes the clinicopathologic features of the reported and personally observed cases of benign mesothelial cells in lymph node. This condition occurs most commonly in the setting of chronic effusion, serosal inflammation, or reaction to adjacent lymphoma. The differential diagnosis from metastatic mesothelioma is discussed.     

自体树突状细胞体外增强肿瘤患者引流淋巴结细胞抗肿瘤活性的作用

目的：探讨自体树突状细胞(DC)体外对胃腺癌患者肿瘤引流区)细胞抗肿瘤活性的增强作用。方法：分离肿瘤患者的外周血单个核细胞(PBMC)经IL-4、GM-CSF和TNF-α诱导其成熟，并以自体肿瘤冻融抗原预致敏，诱导其中的DC。另外，分离胃癌患者的TDLN细胞，在含有IL-2的培养体系中培养，并将TDLN细胞分为3组：(1)第1组为肿瘤抗原致敏的DC加TDLN细胞(D组)；(2)第2组为冻融的肿瘤抗原加TDLN细胞(Ag组)；(3)第3组不加DC及肿瘤抗原作为对照组(L组)。比较3组TDLN细胞对胃腺癌细胞系KATO3和黑色素瘤细胞系A375的杀伤作用。结果：D组对KATO3细胞系的杀伤作用明显优于Ag组与L组；而对A375细胞，各组细胞的杀伤作用没有明显差异。结论：自体DC可明显增强胃癌患者TDLN细胞的杀伤作用。     

Cytochemistry of Reed-Sternberg cells in lymph node imprints

Cytochemical reactions were examined in lymph node imprints from a group of 53 previously untreated patients with histologically proven Hodgkin's disease. In 40 of 51 cases investigated, Reed-Sternberg (R-S) cells, irrespective of the cytologic appearances and the histologic types, showed moderate to strong reactions with acid phosphatase (ACP). In 12 cases ACP activity was present in more than 25% of the R-S cells. The reaction consisted of formation of small- to medium-sized granules, which were located close to the nuclei on a diffusely positive background or irregularly distributed throughout the cytoplasm. In three cases, a coarse granular reaction product with periodic acid-Schiff was present. R-S cells were positive to the naphthol-AS acetate esterase and beta-glucuronidase reactions in four and two cases, respectively. Alkaline phosphatase and naphthol-AS-D-chloroacetate esterase reactions were completely negative. Our results have revealed a pattern of staining in the diagnostic R-S cells similar to that in its morphologic variants; this supports the view that these cells may derive from a common primitive cell. Moreover, the quality and quantity of the ACP reaction product shows that R-S cells differ from both neoplastic histiocytes of malignant histiocytosis and neoplastic lymphocytes of T-cell lymphomas. This study confirms that R-S cells lack definite cytochemical characteristics of each of supposed progenitor cells: histiocytes and T-lymphocytes.     

Afferent lymph and lymph borne cells: their influence on lymph node function

In AO rats the afferent lymphatics to the right cervical lymph nodes (LN) were interrupted and the LN were encased in silicone rubber tubes to prevent reunion of the lymphatics. At regular intervals over the next 12 weeks the following were measured in comparison with the intact contralateral LN - LN weight, influx of lymphocytes from the blood, blood flow, the incorporation of 125IUdR and the incorporation of 35S-sulphate into high endothelial venules (HEV). Systematic histological observations are also reported. One day after deafferentization lymphocyte influx was significantly reduced although blood flow was unchanged and a temporary increase in LN weight was associated with crowding of the lymphatic sinuses with small lymphocytes. The subsequent decline in lymphocyte influx was biphasic and quicker than the decline of other parameters--being undetectable by 6 weeks. Flattening of HEV and diminished secretion of 35S-sulphate was noted at 1 week and progressive degeneration and eventual disappearance of the HEV network was seen by 6-12 weeks. Doubtlessly because of lack of antigenic stimulation 125IUdR incorporation, and numbers of lymphoblasts, plasma cells and finally germinal centres were progressively reduced. The numbers of macrophages and interdigitating cells (IDC) were greatly reduced by 3 weeks and very few were present at 6 weeks probably because most or all arrive in afferent lymph and have a limited life span in the LN. At 12 weeks the LN was difficult to recognize as such since only stromal cells and occasional small lymphocytes remained. In supplementary experiments u.v. irradiation of the LN at the time of deafferentization reduced lymphocyte influx without affecting blood flow suggesting that a u.v. sensitive cell like the IDC may influence lymphocyte influx. In conclusion the involution of the deafferentized LN is partly due to the lack of antigen but progression to the complete loss of specialized structure and function is probably due to lack of other factors including non-lymphoid cells that normally arrive in afferent lymph.     

Electrophoretic mobilities of antigen-stimulated lymph node cells

The effect of immunization against a bacterial antigen on electrophoretic mobility of lymph node cells has been studied.

Incubation of non-stimulated lymph node cells with antigens altered their electrophoretic mobility. However, on washing the antigen away, the original mobility was regained.

Antigen-stimulated lymph node cells had a lower electrophoretic mobility. It was further irreversibly reduced on incubation with the same antigen. This suggests the possibility of formation of firm antigen—antibody complexes at the cell surface.

Implications of these findings and feasibility of using the technique of cell electrophoresis for the study of immune response at the cellular level is discussed.

     

Constitutive and inducible expression of interleukin-6 by Langerhans cells and lymph node dendritic cells

During the induction phase of contact sensitization and other cutaneous immune responses a proportion of epidermal Langerhans cells (LC) is induced to leave the skin and migrate via afferent lymphatics to lymph nodes draining the site of exposure. The cells that accumulate in draining nodes have acquired the characteristics of immunostimulatory dendritic cells and effectively present antigen to responsive T lymphocytes. In the present study we have questioned whether LC in the epidermis and the lymph node dendritic cells into which they develop express interleukin-6 (IL-6), a cytokine that has been shown to serve as an important costimulator of T lymphocyte activation. In situ immunocytochemical analyses using a biotin–streptavidin staining technique revealed that dendritic cells resident in the epidermis of untreated mice constitutively express this cytokine. Keratinocytes expressed detectable IL-6 only following local exposure to the contact allergen oxazolone. Such treatment also appeared to enhance the expression by epidermal dendritic cells of this cytokine. Analyses of unfractionated and LC-enriched and -depleted populations of epidermal cells revealed a close correlation between major histocompatibility complex (MHC) class II (Ia) antigen expression and staining for IL-6, implicating LC as the sole or major source of this cytokine in unstimulated epidermis. Finally, compared with tissue isolated from mice treated with vehicle alone, draining lymph nodes prepared from animals 18 hr following sensitization with oxazolone displayed a substantial increase in both the frequency of dendritic cells and the number of IL-6⁺ cells within the paracortex. These data demonstrate that resident epidermal LC and the dendritic cells into which they develop are important sources of IL-6. Their constitutive and inducible expression of this cytokine will facilitate the induction of cutaneous immune responses.     

Antigen-specific proliferation of murine lymph node cells in vitro

Summary A method is described for determining the antigen-specific proliferative response of murine cells to foreign antigens in vitro. Methods are given for immunizing mice and for preparing lymphoid cells. Culture of lymphoid cells with antigen and quantitation of the proliferative response by determination of the uptake of tritiated thymidine is described. Applications of this methodology are indicated.