Lymphocyte responses of human neonates to bacterial antigens

Human lymphoid lines derived from normal or neoplastic B cells were assayed for insulin binding. ¹²⁵I-Labeled insulin was allowed to bind to cells. Bound radioactivity which was inhibited with unlabeled insulin was regarded as specific binding. Among 46 lines tested, 43 bound more insulin than normal peripheral B lymphocytes. The majority of the lines resembled activated lymphocytes, with regard to their insulin binding. More mature cells represented by EBV-transformed lines of normal origin, bound more insulin than the less differentiated Burkitt lymphoma lines. However, even the latter bound significantly more insulin than peripheral blood lymphocytes.     

Immune islet killing mechanisms associated with insulin-dependent diabetes: in vitro expression of cellular and antibody-mediated islet cell cytotoxicity in humans

In previous work we have shown that some bacteria can bind to human lymphocytes and can be used to identify lymphocyte subpopulations in conventionally stained blood smears. These bacteria are of different species or genera, which makes it difficult to study the binding mechanism. Also, the main marker for B cells, Brucella melitensis, is of very small size and highly pathogenic. Here we show that B cells as well as some of the T cell subpopulations can be identified by different mutants obtained from a strain of an Escherichia coli. Two procedures were used to generate mutants. First, E. coli-YS57 (pro-his-trp-) was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine and the binding to mouse spleen cells was used as a selective pressure. Second, phage-resistant mutants of E. coli-YS57 were obtained and tested for the ability to bind to lymphocytes. Out of 10 strains selected by the former procedure, 5 bound to a significant number of human lymphocytes. All four phage-resistant mutants bound to human lymphocytes. Out of the total of nine mutants that bound to lymphocytes, six bound consistently, i.e., they bound to similar percentages of peripheral blood lymphocytes from different normal donors. One phage-resistant mutant, E. coli USC-106, bound only to B cells. The subpopulations of lymphocytes identified by the mutants were essentially the same as those identified by different species or genera of bacteria. We concluded that E. coli mutants can be obtained that identify human lymphocyte subpopulations and that one of these mutants recognizes B cells; these mutants may be used to study the nature of the receptors for bacteria on lymphocytes, which appear to have a lectin-like nature.     

CD44 and hyaluronan-dependent rolling interactions of lymphocytes on tonsillar stroma

Little is known about how lymphocytes migrate within secondary lymphoid organs. Stromal cells and their associated reticular fibers form a network of fibers that radiate from high endothelial venules to all areas of the lymph node and may provide a scaffold for lymphocyte migration. We studied interactions of lymphocytes with cultured human tonsillar stromal cells and their extracellular matrix using shear stress to distinguish transient interactions from firm adhesion. Tonsillar lymphocytes and SKW3 T lymphoma cells tethered and rolled on monolayers of cultured tonsillar stromal cells and their matrix. A significant proportion of these rolling interactions were independent of divalent cations and were mediated by CD44 binding to hyaluronan, as shown by inhibition with mAb to CD44, soluble hyaluronan, as hyaluronidase treatment of the substrate, and O-glycoprotease treatment of the rolling cells. O-glycoprotease treatment of the substrate also blocked binding completely to stromal matrix and partially to stromal monolayers. SKW3 cells tethered and rolled on plastic-immobilized hyaluronan, confirming the specificity of this interaction. By contrast, monolayers of resting or stimulated human umbilical vein endothelial cells failed to support CD44- and hyaluronan-dependent rolling. SKW3 cells added under flow conditions to frozen sections of human tonsil bound and rolled along reticular fibers in the presence of EDTA. Rolling was blocked by either CD44 mAb or hyaluronan. We propose that lymphocytes migrating through secondary lymphoid organs may use CD44 to bind to hyaluronan immobilized on stromal cells and reticular fibers.     

Insulin complex binding to human peripheral and mitogen-stimulated lymphocytes

Surface labeling and internalization of insulin was demonstrated ultrastructurally with human peripheral lymphocytes and with "activated"/transformed lymphocytes from mitogen-treated cultures using the colloidal gold-labeled insulin-bovine serum albumin (GIA) procedure. The majority of peripheral lymphocytes bound only limited amounts of the insulin complex, while approximately 15% of the lymphocyte population bound modest to comparatively large quantities of the labeled hormone. Quantitative labeling data indicated a skewed GIA labeling continuum for peripheral lymphocytes rather than separate, distinct populations. Sequential labeling studies with the GIA complex followed by either the ferritin-conjugated goat anti-human immunoglobulin or the E-rosette techniques indicated that insulin labeling was neither T nor B cell specific, since extremes of GIA labeling were found in both populations. Many, but not all, circulating lymphocytes with elevated insulin binding had morphological features suggestive of "active" cells, viz., larger cell, nuclear, nucleolar, and Golgi sizes, dispersed chromatin, and greater numbers of polysomes than lymphocytes having minimal GIA labeling. Both phytohemagglutinin (PHA), a T-cell mitogen, and pokeweed mitogen (PWM), a B/T cell mitogen, induced an increase in mean GIA labeling of cultured lymphoid cells as compared to non-mitogen-treated controls. The majority of mitogen-transformed "blast-like" cells had more extensive insulin labeling than nontransformed small (medium)-size lymphocytes, although an overlap in labeling densities was noted in these two groups. PHA induced a slight increase in mean surface GIA labeling of the nontransformed lymphocyte population at 48 and 72 hr of culture as compared to similar cells in non-mitogen-treated controls and PWM cultures. We interpret these findings as indicating the emergence of increased numbers of insulin binding sites on lymphocytes, both those in the circulation and in mitogen-treated cultures, during the early response (activation) to functional and/or metabolic modulations of the cell; this surface change does not appear to be directly related to blastogenic transformation.     

EMMA-4 analysis of iron in cells of the thymic cortex of a weaver-bird (Quelea quelea).

Combined morphological and analytical studies with the EMMA-4 analytical electron microscope have enabled very early erythroid cells to be identified within the cortex of enlarging thymic lobes of Quelea quelea. These early erythroid cells have pale cytoplasm (sometimes with ferritin-like crystals present), slightly pachychromatic nuclei and have fewer cell organelles (mitochondria) than lymphocytes. Counts for iron were approximately 70% lower than counts from mature erythrocytes found free in the cortex. Iron was also recorded from some epithelial reticular cells and pyknotic nuclei; no iron was recorded from small lymphocytes (thymocytes) in the cortex. The presence of very early erythroid cells is a further indication that erythropoiesis occurs in situ in the avian thymus.     

Receptor mobility and the binding of cells to lectin-coated fibers

The ability of cells to bind to nylon fibers coated with lectin molecules interspaced with varying numbers of albumin molecules has been analyzed. The cells used were lymphoma cells, normal lymphocytes, myeloid leukemia cells, and normal and transformed fibroblasts, and the fibers were coated with different densities of concanavalin A or the lectins from soybean or wheat germ. Cells fixed with glutaraldehyde did not bind to lectin-coated fibers. The number of cells bound to fibers could be increased by increasing the density of lectin molecules on the fiber, the density of specific receptors on the cell, or the mobility of the receptors. It is suggested that binding of cells to fibers involves alignment and binding of specific cell surface receptors with lectin molecules immobilized on the fibers, and that this alignment requires short-range rapid lateral mobility (RLM) of the receptors. The titration of cell binding to fibers coated with different densities of lectin and albumin has been used to measure the relative RLM of unoccupied cell surface receptors for the lectin. The results indicate a relationship of RLM to lectin-induced cell-to-cell binding. The RLM or receptors for concanavalin A (Con A) was generally found to be higher than that of receptors for the lectins from wheat germ or soybean. Receptor RLM could be decreased by use of metabolic inhibitors or by lowering the temperature. Receptors for Con A had a lower RLM on normal fibroblasts than on SV40-transformed fibroblasts, and trypsinization of normal fibroblasts increased Con A receptor RLM. Normal lymphocytes, lymphoma cells, and lines of myeloid leukemia cells that can be induced to differentiate had a high receptor RLM, whereas lines of myeloid leukemia cells that could not be induced to differentiate had a low receptor RLM. These results suggest that the RLM of Con A receptors is related to the transformation of fibroblasts and the ability of myeloid leukemia cells to undergo differentiation     

Identification of Ia on a subpopulation of human T lymphocytes that stimulate in a mixed lymphocyte reaction

The delineation of discrete subpopulations of human T lymphocytes has permitted preliminary analyses of the complex cellular network regulating the immune response in man. We previously showed that a subset of T lymphocytes, designated as theophylline-sensitive because of their inability to bind sheep red blood cells in the presence of the drug, are responsible for antigen-specific suppression or regulation in an in vitro plaque-forming cell assay. We now show that 25 to 45% of these theophylline-sensitive T cells were Ia-positive by immunofluorescence with a rabbit antiserum raised against purified B lymphoblast surface antigenic material. These data suggested that 4 to 7% of peripheral blood T cells carry Ia determinants. The presence of Ia determinants on this T cell subset was confirmed by gel analysis of radioiodinated surface material. Furthermore, in mixed lymphocyte culture, the theophylline-sensitive cells demonstrated HLA-D determinants and were 10-fold more potent stimulators than equal numbers of B lymphocytes. The presence of Ia determinants on these T cells indicates the expression of major histocompatibility complex-related regulatory gene products on a specific human T lymphocyte subpopulation.     

Subpopulations of B cells in germinal centers. III. HJ6, a monoclonal antibody, binds globoside and a subpopulation of germinal center B cells.

To identify surface Ag uniquely expressed on human germinal center B cells, we produced a mouse mAb, HJ6. When tonsillar lymphocytes were examined, HJ6 did not label T cells and labeled only about half of PNA+ B cells that were HK23-. HJ6 did not label mononuclear cells from peripheral blood, splenocytes, and any of 29 cell lines including 23 B cell lines. This binding pattern of HJ6 was very similar to that of a mAb named 5B5. It was shown previously that 5B5 bound a glycolipid named CTH (CD77) and its Ag was expressed on HK23- PNA+ tonsillar lymphocytes and Burkitt's lymphoma cell lines. Despite the similarity, HJ6 differed from 5B5: HJ6 did not stain Burkitt's lymphoma cell lines and stained PNA+ tonsillar lymphocytes in the presence of a large concentration of galactose. When its binding to isolated glycolipids was studied, HJ6 was found to bind globoside and Forssman Ag and not to other glycolipids including CTH. When its binding to neutral glycolipids extracted from tonsillar lymphocytes was studied, HJ6 bound only globoside; Forssman Ag was not detected in tonsillar lymphocytes. Taken together, we conclude that globoside is a B cell Ag expressed on a subpopulation of germinal center B cells.     

Persistence of lpr/lpr-derived IgG2a secreting B cells in normal----lpr/lpr adult radiation chimeras or lpr/lpr----normal kidney capsule chimeras.

Lethally irradiated MRL/lpr mice reconstituted with bone marrow stem cells from a normal mouse strain develop a state of split hematopoietic chimerism; erythrocytes, granulocytes, and macrophages are derived from the normal stem cell inoculum while the peripheral T lymphocytes are derived from radioresistant lpr host cells. Moreover, these mice have normal levels of serum IgM and IgG2a produced by radioresistant host B cells, even though they have relatively few sIgM+ B cells. In order to better understand the differentiation and regulation of B cells present in these chimeric mice, the current study was undertaken to localize and to assess the functional capacity of the lpr B cells producing the serum antibodies. Surface IgG2a+ cells could not be found in the spleen or lymph nodes of these mice, but large lymphocytes containing cytoplasmic IgG2 of host (lpr) allotype could be readily detected, even though they constituted less than 1% of the total spleen population. The host-derived serum IgG2 and IgG2+ cells were even present in the spleens of "leaky" mice that had relatively normal numbers of donor-derived sIgM+ B cells. These lpr B cells secreted IgG2a antibody that bound ssDNA, but they could not respond to immunization with SRBC. These results indicate that the lpr-derived radioresistant B cells have a limited capacity for proliferation and are already committed to the memory lineage. The presence of similar B cells in normal mice transplanted with neonatal lpr/lpr spleen fragments suggests that lpr/lpr B cell development is inherently abnormal.     

Ontogeny of lymphocytes expressing J chain in chickens

The ontogeny of chicken lymphocytes expressing J chain (LEJ) was investigated in the embryonic bursa of Fabricius, the spleen, and the thymus. Simultaneous appearance of LEJ was detected in the bursa and spleen on Day 14 of incubation. These cells were detected later in the thymus. The LEJ were found to increase rapidly in the spleen from the 19th to 20th incubation day. In adult chickens, the highest percentage of LEJ was also found in the spleen. These cells were seen in the thymus at a lower frequency. Intermediate numbers were found in bursal and peripheral blood lymphocytes. The frequencies of the LEJ were similar to those of lymphocytes positive for cytoplasmic immunoglobulins (Ig) IgA and IgM, but were not related to the number of lymphocytes expressing surface Ig. It is possible to consider that the suitable site for LEJ is the spleen, on the basis of the rapid increase in the number of LEJ just before hatching and from the fact that the highest value is found in adult chickens. Furthermore, LEJ may participate in secretion of IgA or IgM but not be associated with the expression of surface Ig.     

Expression of low levels of peripheral lymph node-associated vascular addressin in mucosal lymphoid tissues: possible relevance to the dissemination of passaged AKR lymphomas

Lymphoid tumors display a wide variety of growth patterns in vivo, from that of a solitary extralymphoid tumor, to a general involvement of all lymphoid organs. Normal lymphocytes are uniquely mobile cells continuously recirculating between blood and lymph throughout much of their life cycle. Therefore, it is reasonable to propose that disseminating malignant lymphocytes may express recirculation characteristics or homing properties consistent with that of their normal lymphoid counterparts. Trafficking of lymphocytes involves the expression and recognition of both lymphocyte homing receptors and their opposing receptors on endothelium, the vascular addressins. These cell surface elements direct the tissue-selective localization of lymphocyte subsets in vivo into organized lymphoid organs and sites of chronic inflammation where specific binding events occur between lymphocytes and the endothelium of specialized high endothelial venules (HEV). In a recent murine study of 13 lymphoma lines, we found that lymphomas that bind well to high endothelial venules, in the Stamper-Woodruff in vitro assay (an assay of lymphocyte binding to venules in frozen sections of peripheral lymph nodes or Peyer's patches), spread hematogenously to all high endothelial venule bearing lymphoid organs, whereas non-binding lymphomas did not. In some cases lymphomas that bound with a high degree of selectivity to peripheral lymph node (PLN) high endothelial venules exhibited only limited organ preference of metastasis, involving the mucosal lymphoid organs Peyer's patches (PP) in addition to the peripheral lymph nodes of adoptive recipients. Here we demonstrate that Peyer's patch high endothelial venules express a low but functional level of peripheral lymph node addressin (MECA-79) that can be recognized by lymphomas expressing the peripheral lymph node homing receptor (MEL-14 antigen).(ABSTRACT TRUNCATED AT 250 WORDS)     

Trypanosoma lewisi: electron microscopy of the infected spleen

The spleens of Lewis rats, both normal and infected with Trypanosoma lewisi were examined by electron microscopy. Special attention was directed to clusters of splenic cells which occur in the course of the infection. The reticular cells first showed alterations of their structure by the second day of infection, with considerable surface membrane activity. By the fourth day and thereafter various cells were found gathered around the reticular cells. These cell clusters mainly contained lymphocytes, plasma cells, and erythropoietic elements in many stages of differentiation. It was not unusual that several cell types were found adjacent to the same central reticular cell. These arrays, similar in geometry to the erythropoietic island of the bone marrow, became more predominantly “plasma cell” islands as the infection progressed. Parasites were recognizable within the reticular cells, and were noted to be in regions where the cellular membranes of adjacent cells demonstrated vesiculations resembling rhopheocytosis. A further observation was the pinching off of neighboring plasma cell cytoplasm into the reticular cells.     

Human lymphocyte responses are enhanced by culture at 40 degrees C.

In vitro responses of human peripheral blood lymphocytes (PBL) were found to be markedly enhanced by culture at 40 degrees C rather than at the conventional temperature of 37 degrees C. We studied proliferative responses of lymphocytes by activation by phytohemagglutinin (PHA) concanavalin A (Con A), pokeweed mitogen (PWM), and allogeneic lymphocytes in mixed lymphocyte culture (MLC) and found enhancement of DNA synthesis at the higher temperature. Cytotoxic T cell responses to allogeneic cells were also enhanced when MLC was done at 40 degrees C. These enhanced immune responses appear to be due in part to increased numbers of participating cells. If in vitro lymphocyte responses correlate with in vivo responses, then fever associated with infection or tumor may be beneficial whereas that associated with autoimmune disorders may have a detrimental effect.     

Characterization of T and B antigen-binding cells for beta-galactosidase. II. T antigen-binding cells.

The results reported in this paper suggest that the specific Z-binding cells of the normal mouse include a large portion of T lymphocytes. Depletion of T cells with anti-theta serum and cortisone indicates that the majority of the ZBC of the thymus, which occur at frequencies of about 150/10(6), are indeed T cells. Similar treatment of spleen cells suggests that approximately half the binding cells in that organ are contributed by the T lymphocyte population. T-and B-enriched populations obtained from the spleen by using differential adherence to nylon wool contained equal numbers of ZBC and bound equivalent amounts of the antigen. Hence, there appears to be a high frequency of T lymphocytes that can be shown to bind beta-galactosidase specifically in both the thymus and spleen of normal mice.     

Human lymphocyte-high endothelial venule interaction: organ-selective binding of T and B lymphocyte populations to high endothelium

We wished to determine whether human lymphocytes, like their murine counterparts, show organ-specific interactions with high endothelial venules (HEV). Functional HEV-binding ability was measured by an in vitro assay of lymphocyte adherence to HEV in frozen sections of human lymphoid tissues which was adapted from rodent systems. It was found that human lymphocytes bind selectively to HEV and that, whereas mature T lymphocytes bind preferentially to HEV in peripheral lymph nodes and tonsils, B lymphocytes show preferential binding to HEV in GALT. Moreover, by analyzing the binding characteristics of T4+ and T8+ T cell populations, it was found that T8+ cells adhere preferentially to HEV in GALT and mesenteric lymph nodes and tonsil, and that T4+ cells bind slightly better to HEV in peripheral lymph nodes. The above findings indicate that organ--specific lymphocyte-endothelial cell recognition mechanisms exist also in humans, and suggest that these mechanisms play an important role in normal and pathologic lymphocyte traffic.     

Accumulation of mucosal T lymphocytes around epithelial cells after in vitro infection with Cryptosporidium parvum.

We had previously shown that ileal intraepithelial lymphocytes isolated from calves with cryptosporidiosis include significantly increased numbers of CD8+ T lymphocytes and activated CD4+ cells. These increases could result from redistribution of resident mucosal lymphocytes or from homing of peripheral T cells to ileal mucosa. To determine whether resident mucosal lymphocytes can redistribute to Cryptosporidium parvum-infected epithelium, oocysts were inoculated in vitro onto ileum explants taken from 1-2-wk-old noninfected calves. After 24 hr of incubation, the explants were collected and frozen in liquid nitrogen. Immunohistochemical analysis of T-lymphocyte subpopulations was performed on sections, and labeled lymphocytes adjacent to villous epithelial cells were counted. Compared with uninoculated explants, there was a statistically significant increase in the number of CD8+ T lymphocytes per 100 epithelial cells in oocyst-inoculated tissue. In addition, there were increased numbers of CD4+ T cells and activated (CD25+) lymphocytes adjacent to C. parvum-infected epithelium. These results show that resident mucosal T lymphocytes can accumulate at the epithelium during C. parvum infection.     

Mechanism of cell-mediated cytotoxicity at the single cell level. VI. Direct assessment of the cytotoxic potential of human peripheral blood non-lytic effector-target cell conjugates

Single cell cytotoxicity assays reveal that a large percentage of lymphocytes are unable to kill attached targets in a 4- to 18-hr assay. Additional signals (in the form of lectin or anti-target antibody) delivered to target-bound lymphocytes enable these previously non-lytic lymphocytes to kill attached target cells. This finding was obtained by using a modification of the single cell assay, in which lectin or target cell antibody is incorporated into agarose with preformed lymphocyte-target conjugates. Human peripheral blood lymphocytes (PBL) or Percoll density gradient-enriched large granular lymphocytes (LGL) were used as effector cells in natural killer (NK), antibody-dependent cellular cytotoxicity (LDCC) assay systems. The targets used were NK-sensitive K562 and Molt-4 and NK-insensitive Raji. Several findings were made in the modified single cell assay, namely a) the frequency of cytotoxic NK or ADCC effector cells was not augmented, suggesting that the initial trigger was sufficient for lytic expression in these instances. Furthermore, these results showed that the NK-sensitive targets used do not bind nonspecifically to the LDCC effector cells. K562 coated with Con A, however, serve as LDCC targets. b) The frequency of two target conjugate lysis by NK/K effectors was not augmented by Con A. These results suggest that Con A does not potentiate the killing of multiple targets bound to a single cytotoxic lymphocyte. c) Although conjugates formed between LGL or PBL and NK-insensitive Raji are non-lethal, significant lysis was observed when these conjugates were suspended in Con A or antibody agarose. These results demonstrate that Raji bind to cytotoxic NK, K, and LDCC effector cells, but are lysed only when the appropriate trigger is provided. d) The cytotoxic potential of non-lytic conjugates appears to lie within the low density Percoll fraction, although the high density lymphocytes are able to nonlethally bind to targets. Altogether the results demonstrate that target recognition and/or binding by the effector cells is a distinct event from the trigger or lytic process. The implications of these findings are discussed.     

PEPTIDASE DISTRIBUTION IN LYMPHATIC TISSUE

The lymphatic tissue of the rabbit contains a labile peptidase as measured by the hydrolysis of alanylglycine. Some characteristics of the enzyme were determined. This enzyme increases in amount when the numbers of macrophages in the tissue are increased and it is also present in the extracellular fluid in high concentration. The extracellular fluid value for this activity is calculated to be about 8 times the value for serum. Based on a correlation between the types of cells present and the amount of peptidase found in the tissue the following relative activities are assigned to the tissue components per unit volume: lymphocytes 1.0, tissue fluid 11.0, serum 1.4, phagocytes (macrophages) 30.0, reticular cells 12.0. The amount of chloride space varied from 35 to 55 per cent. The relative amounts of acid phosphatase per unit volume in the same elements were calculated to be: lymphocytes 1.0, tissue fluid 0, phagocytes 20.0, and reticular cells 4.0. Analysis of the distribution of peptidase was facilitated by simultaneous determination of acid phosphatase whose primary localization in one cell type was known. The over-all contribution of lymphocytes to the labile peptidase content of lymphatic tissue is relatively minor and was not found to exceed 5 per cent of the average value for the entire nodular tissue. In the absence of large numbers of macrophages the intercellular fluid of the nodule accounts for half or more of the peptidase content of the nodules.     

Binding of acridine orange by chromatin in human lymphocytes with different capabilities for adhesion]

A population of morphologically homogenous peripheral blood lymphocytes separated on a column with glass beads was studied microfluorimetrically. It was found that high adhesive lymphocytes bound acridine orange in a greater extent than did low adhesive cells.     

P-selectin (CD62) binds to subpopulations of human memory T lymphocytes and natural killer cells.

P-selectin (CD62) is a Ca(2+)-dependent lectin expressed on activated platelets and endothelium. Although P-selectin is known to function as a receptor for myeloid cells, previous studies indicated that P-selectin also bound to a subset of lymphocytes. Using a multi-color immunofluorescence assay we found that purified P-selectin bound to 12.2 +/- 4.1% of peripheral blood lymphocytes and that P-selectin could mediate adhesion of activated platelets to lymphocytes. A subpopulation of CD4+, CD8+, and CD16+ lymphocytes bound P-selectin. There was a marked preference for P-selectin binding to memory cells (CD45RO+) in both the CD4+ and CD8+ populations. Binding to all cell types was Ca(2+)-dependent and blocked by pretreatment of the cells with sialidase. These data suggest that P-selectin may play a role in the recruitment of specific lymphocyte populations to sites of inflammation.