Frequency analysis of human peripheral blood B cells producing autoantibodies: differential activation requirements of precursors for B cells producing IgM-RF and anti-DNA antibody.

To investigate the mechanism of autoantibody production, the extent and nature of anti-DNA precursors within normal human B cells were examined by utilizing two different polyclonal B cell stimulators, Staphylococcus aureus Cowan I (SA) and immobilized mAb to the CD3 molecular complex (64.1). In cultures stimulated with SA and IL2, B cells produced IgM-RF, but not anti-DNA, whereas B cells produced great amounts of anti-DNA in cultures stimulated with SA and intact T4 cells. In cultures stimulated with immobilized anti-CD3, T4 cells that had been treated with mitomycin C (T4 mito) induced the production of anti-DNA as effectively as that of IgM-RF. Limiting dilution analyses revealed that the precursor frequency of anti-DNA-producing cells in SA-stimulated cultures was markedly increased in the presence of intact T4 cells (0.399 to 2.549 per 10(4) B cell) compared with that in the presence of factors generated from mitogen-activated T cells (TF) (0.022 to 0.151 per 10(4) B cells). Thus, the proportion of IgM-secreting cells that produced anti-DNA in cultures with SA + T4 cells (12 to 31%) was much greater than that noted in cultures with SA + TF (3.4 to 4.0%), whereas the proportion of IgM-secreting cells that produced IgM-RF was not significantly different in these cultures (17 to 50%). The precursor frequency of anti-DNA-producing cells (0.019-0.097 per 10(2) B cells) was almost the same as that of IgM-RF producing cells (0.025-0.104 per 10(2) B cells) in cultures stimulated with immobilized anti-CD3. Of note, addition of SA increased the precursor frequency of IgM-RF-producing cells, but not that of anti-DNA-producing cells, in anti-CD3-stimulated cultures. These results indicate that T cells, but not T cell-derived cytokines, play a central role for the production of anti-DNA antibodies. Moreover, the data support the conclusion that the precursors of anti-DNA-producing cells have activation requirement different from those of IgM-RF producing cells.     

Cytotoxicity of effector cells of the peripheral blood, lymph nodes and spleen in Hodgkin's disease

The mononuclear cells and T-lymphocytes of the blood, spleen and lymph nodes from 83 patients with Hodgkin's disease and 50 healthy donors were tested in assays for lectin-dependent (LD) and natural killer (NK) cytotoxic activity (CTA). On an average, peripheral blood T cell LD-CTA of patients did not differ from that of the donors. However, the CTA appeared to be dependent on the stage of the disease; in the IVth stage LD-CTA was decreased 2-fold. The LD-CTA was also dependent on the histological type of disease and the lowest level of LD-CTA (50% of the control level) was associated with the "lymphocyte depletion" type. The CTA of T-lymphocytes from the affected areas of the patients' spleen was more marked than that of the unaffected areas. Spleen cell CTA showed no other correlations. The CTA of lymphocytes from the affected lymph nodes was drastically lower than CTA of blood and spleen lymphocytes. The NK activity of the patients' blood and spleen lymphocytes was twice as less as the control level (healthy donors) and did not correlate with a stage and/or a histological type of the disease. It was assumed that in Hodgkin's disease the specific antitumor immunity remains mostly within normal and is decreased only in the last, terminal stage of the disease.     

Lymph node, spleen and peripheral blood lymphocytes as stimulators of alloreactivity

Before and after kidney transplantations, in vitro tests that measure the level of reactivity between donor and recipient lymphocytes are performed for better organ selection and as indicator of possible organ rejection. In these tests, donor's and recipient's lymphocytes are stimulated for proliferation, which intensity is measured and accordingly organ recipient reactivity towards graft is determined. Lymph node, spleen and peripheral blood lymphocytes are used for those purposes. For better interpretation of these in vitro tests it should be important to determine mitogenic ability of lymphocytes of different origin and to choose the most adequate cells. To compare mitogenic ability of deceased donor lymph node, spleen and peripheral blood lymphocytes one-way mixed lymphocyte culture (MLC) was used. As stimulators irradiated lymphocytes from spleen, lymph node and peripheral blood samples of 12 deceased donors were used while as responders lymphocytes from peripheral blood of healthy individuals, chosen according HLA-DRB1 alleles (stimulators and responders were HLA-DRB1 identical, semi-identical or different), were used. Spleen lymphocyte activity was the best with different cells and the weakest with identical cells. Impact of polyclonal mitogens (PHA - phytohemagglutinin, Con A - concanavalin A and PWM - pokeweed mitogen) on lymphocyte proliferation was tested on lymphocytes from spleen and lymph node of deceased donors. Results obtained in culture in vitro showed that spleen cells had exerted the best mitogenic potential and PHA had the greatest impact upon lymphocyte proliferation. This investigation is of importance for establishing the best model to reflect in vivo situation in transplanted patient.     

Differential response of B cells in the lymph node and the spleen to bacterial lipopolysaccharide

In vivo polyclonal activation of B cells in the lymph nodes and the spleens of mice injected with bacterial lipopolysaccharide (LPS) was compared. The peak of anti-trinitrophenylated sheep red blood cells plaque-forming cell (PFC) response in the lymph node was reached 6-8 days after the injection of LPS while that in the spleen was reached at 2 days. The maximal increase in the total number of Ig-producing cells in the lymph node also occurred at the later stage. These differences in time courses of polyclonal activation of B cells between the lymph node and the spleen were not due to the absence of B cells in the lymph node, migration of PFC from the spleen to the lymph node, or qualitative differences of B cells. This phenomenon was dependent on the environmental difference between the lymph node and the spleen, because B cells from the lymph node could respond to LPS rapidly in the spleen. Further, the polyclonal activation of B cells was accelerated in the lymph nodes of mice receiving prior injection of LPS. In in vitro cultures of lymph node cells of those mice, a significant amount of interleukin-1 could be detected by stimulation of LPS. It was possible that the delayed activation of B cells in the lymph node was due to the time lag necessary for construction of the environmental condition suitable for activation of B cells, whereas in the spleen this condition can be provided without delay.     

Plaque forming cells in the spleen,lymph and blood

The capacity of blood and lymphatic cells and of the spleen in rabbits immunized with sheep red blood cells to produce antibodies (PFC) has been studied. In rabbits in which only the blood was examined, the numbers of PFC reached a peak 4 days after one immunization, or 3 days after two immunizations. Moreover, double immunization yielded 10 times higher numbers of PFC than single immunization. Comparative studies of the blood, lymph and spleen revealed largest numbers of PFC in the spleen, smaller in the blood, and fewest in the lymph. This pattern was observed after single as well as double immunization, although the proportions were different after double immunization. After the second immunization much larger numbers of PFC appeared, increasing especially in the lymph. Discrepancies between the findings and earlier results, and the problem of the mechanisms of permeation of immunologically competent cells into the blood are discussed.     

The human peripheral lymph node vascular addressin is a ligand for LECAM-1, the peripheral lymph node homing receptor

The trafficking of lymphocytes from the blood and into lymphoid organs is controlled by tissue-selective lymphocyte interactions with specialized endothelial cells lining post capillary venules, in particular the high endothelial venules (HEV) found in lymphoid tissues and sites of chronic inflammation. Lymphocyte interactions with HEV are mediated in part by lymphocyte homing receptors and tissue-specific HEV determinants, the vascular addressins. A peripheral lymph node addressin (PNAd) has been detected immunohistologically in mouse and man by monoclonal antibody MECA-79, which inhibits lymphocyte homing to lymph nodes and lymphocyte binding to lymph node and tonsillar HEV. The human MECA-79 antigen, PNAd, is molecularly distinct from the 65-kD mucosal vascular addressin. The most abundant iodinated species by SDS-PAGE is 105 kD. When affinity isolated and immobilized on glass slides, MECA-79 immunoisolated material binds human and mouse lymphocytes avidly in a calcium dependent manner. Binding is blocked by mAb MECA-79, by antibodies against mouse or human LECAM-1 (the peripheral lymph node homing receptor, the MEL-14 antigen, LAM-1), and by treatment of PNAd with neuraminidase. Expression of LECAM-1 cDNA confers PNAd binding ability on a transfected B cell line. We conclude that LECAM-1 mediates lymphocyte binding to PNAd, an interaction that involves the lectin activity of LECAM-1 and carbohydrate determinants on the addressin.     

Priming of peripheral lymph node B cells with TNP-Ficoll: role of lymphokines in B cell differentiation.

We have previously shown that peripheral lymph node (PLN) B lymphocytes of adult DBA/2J mice failed to make an antibody response to type 2 antigen TNP-Ficoll, but exhibited a good antibody response to type 1 antigen TNP-Brucella abortus. In the present study we wanted to find out whether the unresponsiveness of PLN B cells to TNP-Ficoll is due to defects in the early activation and proliferation stage or in the final differentiation stage of B cells. Therefore, we have used a two-step protocol of in vivo immunization of mice with TNP-Ficoll and the subsequent in vitro challenge with TNP-Brucella abortus and studied the anti-TNP plaque-forming cell (PFC) responses. The results indicate a three- to sixfold increase of PFC responses in PLN cell cultures derived from TNP-Ficoll-primed animals compared to saline control mice. This increased antibody response was TNP-specific as 93% of the PFC's were inhibited by TNP-lysine. Limiting dilution experiments confirm that the increase in anti-TNP PFC response from the TNP-Ficoll-primed animals was indeed due to an increase in TNP-specific precursor B cells. Further, the addition of rIL-5 or rIL-6 induced anti-TNP PFC in the TNP-Ficoll-primed and in control PLN cell cultures in the presence of antigen. However, in primed PLN cells lymphokines alone were sufficient to restore anti-TNP PFC response. In conclusion, our results show that in PLN, the TNP-Ficoll can induce proliferation of hapten-specific B cells but not final differentiation. These primed PLN B cells mature into antibody-secreting cells upon stimulation with TNP-BA or lymphokines.     

The anatomy of peripheral lymphoid organs with emphasis on accessory cells: light-microscopic immunocytochemical studies of mouse spleen, lymph node, and Peyer's patch

Antigen, lymphocytes, and accessory cells interact within peripheral lymphoid organs to generate immunity. Two cell types have been studied for accessory function in culture: mononuclear phagocytes and nonphagocytic Ia-rich dendritic cells. The monoclonal antibodies which have been used to study isolated murine macrophages (M phi) and dendritic cells (DC) include alpha-macrophage (F4/80, M1/70), alpha-dendritic cell (33D1), alpha-Fc receptor (2.4G2), and alpha-Ia (B21-2) reagents. In this paper, the antibodies have been used to stain accessory cells in cryostat sections of mouse spleen, lymph node, and Peyer's patch. Each organ is known to contain subregions that are rich in either macrophages, B cells, or T cells. We found that the accessory cells in each subregion had a different phenotype. 1) Macrophage-rich regions: Macrophages that lined the site of antigen delivery (marginal zone of spleen, around afferent lymphatics of node, and below the epithelium of Peyer's patch) were stained with M1/70 but not with F4/80. F4/80 was abundant on macrophages in other sites: spleen red pulp, node medulla, and around Peyer's patch efferent lymphatics. 2) B-lymphocyte-rich follicles: Follicular dendritic cells, which retain immune complexes extracellularly, are concentrated on the outer aspect of the germinal center. This region stained strongly with alpha-Fc receptor antibody 2.4G2, but not with M1/70, F4/80, or 33D1. 3) T areas: The interdigitating cells of T areas have been linked to isolated dendritic cells. Irregular Ia-rich cells were distributed uniformly in the T areas of each organ. However, staining with 33D1 was not detected and was restricted to foci of nonphagocytic cells at the spleen red/white pulp junction. F4/80, M1/70 or 2.4G2 also did not stain the T area, except for the region close to splenic central arteries. Therefore the principal surface markers and locations of the candidate accessory cells in murine lymphoid organs are M1/70+ macrophages at the site of antigen entry; F4/80+ macrophages around regions of lymphocyte efflux; germinal center dendritic cells, which may be rich in 2.4G2; and Ia-rich interdigiting cells in the T area.     

Ultrastructural analysis of HNK-1+ cells in human peripheral blood and lymph nodes

HNK-1 positive (HNK-1+) cells in human peripheral blood and lymph nodes were comparatively analysed by means of immunohistochemistry and immunoelectron microscopy. In peripheral blood, the HNK-1+ cells were grouped into large granular lymphocytes (LGLs), small lymphocytes and intermediate forms, all of which had many fine cytoplasmic processes. Except for smooth-surfaced lymphocytes, they could not be distinguished from helper/inducer T (OKT4/Leu3a) cells and suppressor/cytotoxic T (OKT8/Leu2a) cells. In double staining, HNK-1+T3- cells and HNK-1+T3+ cells could not be clearly distinguished in terms of morphology, although the former contained many LGLs. The HNK-1+ cells in the lymph nodes accumulated in the light zones of the germinal centers (GCs). These cells were small to medium-sized lymphocytes with few electron-dense granules and exclusively co-expressed helper/inducer T cell antigens (HNK-1+T4+). Their cytoplasmic projections were interwoven with those of the follicular dendritic cells which trap immune complexes for a long duration. These configurations suggest that HNK-1+T4+ cells in GCs are engaged in an immunological regulation of germinal center cells. On the other hand, large blastic HNK-1+ cells were scattered outside the GCs and some of them were in the process of mitosis. Furthermore, HNK-1+LGL-like cells with a few large electron-dense granules were rarely seen. These observations indicate that the HNK-1+ cells in the lymph nodes may proliferate outside GCs and differentiate into LGLs with a strong natural killer function.     

Analysis of lymphoid and dendritic cells in human lymph node, tonsil and spleen. A study using monoclonal and heterologous antibodies

The distribution of lymphoid and dendritic cells in human reactive lymph nodes, tonsils and spleens was examined by means of an indirect immunoperoxidase technique, using a panel of monoclonal and heterologous antibodies. The antibodies used were directed against antigens present on T cell subsets (Leu1, leu2a, Leu3a, TA1, OKT6), various types of B cells (BA1, BA2, HLA-DR, CR1) and cells of the mononuclear phagocyte system (alpha HM1, TA1, CR1, OKM1, NA 1/34). In the lymph node and tonsil Leu3a-positive cells (T-helper/inducer phenotype) and Leu2a-positive cells (T-suppressor/cytotoxic phenotype) are found in the thymus-dependent or T-cell area; in the spleen Leu3a-positive cells are found mostly in the periarteriolar lymphocyte sheath (PALS), while Leu2a-positive T-suppressor/cytotoxic cells are almost completely restricted to the cords of Billroth in the red pulp. The cells in the mantle zone of germinal centres and in the primary follicles in lymph nodes, tonsils and spleens have B-cell properties (BA1-, HLA-DR-, and CR1-positive). The cells in the germinal centres show a similar staining pattern (HLA-DR-, and partly CR1-positive). Follicles and T-cell-dependent areas have specific dendritic cells, each with a specific staining pattern: the dendritic reticulum cell (DRC) of the follicle stain with CR1, HLA-DR, BA2 and alpha HM1; the interdigitating cell of the T-cell areas in the lymph node, tonsil and spleen stain with HLA-DR and BA1. Moreover, large dendritic OKT6-positive cells are found in the T-cell areas of some of the peripheral lymph nodes, and are probably Langerhans cells. It is concluded that human lymph nodes and tonsils have an identical compartimentalisation, clearly differing from the spleen in cellular organization.     

Activation of human B lymphocytes: XVI. Cellular requirements,interactions, and immunoregulation of pokeweed mitogen-induced total-immunoglobulin producing plaque-forming cells in peripheral blood

The optimal culture and assay conditions for the detection of spontaneously occurring and pokeweed mitogen (PWM)-induced polyvalent Ig (IgG + IgM + IgA) and individual Ig class-specific plaque-forming cells (PFC) in human peripheral blood have been described in detail. Culture conditions are critical, particularly with regard to cell density and batches of supplemental serum. Fetal calf serum is a much more supportive serum supplement for PWM-induced PFC than is human serum. The assay system is a modified reverse hemolytic PFC assay using staphylococcal protein A coupled to sheep red blood cells by the chromic chloride method. PFC are developed by rabbit anti-human polyvalent Ig or anti-human individual Ig class antisera. Human peripheral blood contains 468 (±78) spontaneously occurring Ig secreting PFC per 10⁶ lymphocytes at Day 0 and 20,500(± 1971) PWM-induced Ig secreting PFC after 6 days in culture. The response is T-cell dependent; however, T cells can be replaced by a soluble T-cell factor prepared from a 48-hr allogeneic mixed lymphocyte reaction supernatant. The relative dependence on monocytes is a reflection of the culture conditions employed. Under the conditions of round-bottom tubes which promote cell-to-cell contact, depletion of monocytes to 0 to 2% does not result in a diminution of PFC responses. In fact, under such conditions, in certain individuals monocytes are markedly suppressive such that removal of monocytes results in a substantial enhancement of PFC responses. This system is simple and reproducible and should prove extremely useful in the delineation of the mechanisms of B-cell triggering and immunoregulation in normals and in disease states.     

Lactate dehydrogenase-elevating virus replication persists in liver, spleen, lymph node, and testis tissues and results in accumulation of viral RNA in germinal centers, concomitant with polyclonal activation of B cells.

Lactate dehydrogenase-elevating virus (LDV) replicates primarily and most likely solely in a subpopulation of macrophages in extraneuronal tissues. Infection of mice, regardless of age, with LDV leads to the rapid cytocidal replication of the virus in these cells, resulting in the release of large amounts of LDV into the circulation. The infection then progresses into life-long, asymptomatic, low-level viremic persistence, which is maintained by LDV replication in newly generated LDV-permissive cells which escapes all antiviral immune responses. In situ hybridization studies of tissue sections of adult FVB mice revealed that by 1 day postinfection (p.i.), LDV-infected cells were present in practically all tissues but were present in the highest numbers in the lymph nodes, spleen, and skin. In the central nervous system, LDV-infected cells were restricted to the leptomeninges. Most of the infected cells had disappeared at 3 days p.i., consistent with the cytocidal nature of the LDV infection, except for small numbers in lymph node, spleen, liver, and testis tissues. These tissues harbored infected cells until at least 90 days p.i. The results suggest that the generation of LDV-permissive cells during the persistent phase is restricted to these tissues. The continued presence of LDV-infected cells in testis tissue suggests the possibility of LDV release in semen and sexual transmission. Most striking was the accumulation of large amounts of LDV RNA in newly generated germinal centers of lymph nodes and the spleen. The LDV RNA was not associated with infected cells but was probably associated with virions or debris of infected, lysed cells. The appearance of LDV RNA in germinal centers in these mice coincided in time with the polyclonal activation of B cells, which leads to the accumulation of polyclonal immunoglobulin G2a and low-molecular-weight immune complexes in the circulation.     

Immunosurveillance by hematopoietic progenitor cells trafficking through blood, lymph, and peripheral tissues

Constitutive egress of bone marrow (BM)-resident hematopoietic stem and progenitor cells (HSPCs) into the blood is a well-established phenomenon, but the ultimate fate and functional relevance of circulating HSPCs is largely unknown. We show that mouse thoracic duct (TD) lymph contains HSPCs that possess short- and long-term multilineage reconstitution capacity. TD-derived HSPCs originate in the BM, enter the blood, and traffic to multiple peripheral organs, where they reside for at least 36 hr before entering draining lymphatics to return to the blood and, eventually, the BM. HSPC egress from extramedullary tissues into lymph depends on sphingosine-1-phosphate receptors. Migratory HSPCs proliferate within extramedullary tissues and give rise to tissue-resident myeloid cells, preferentially dendritic cells. HSPC differentiation is amplified upon exposure to Toll-like receptor agonists. Thus, HSPCs can survey peripheral organs and can foster the local production of tissue-resident innate immune cells under both steady-state conditions and in response to inflammatory signals.     

Subpopulations of human T lymphocytes. III. Distribution and quantitation in peripheral blood, cord blood, tonsils, bone marrow, thymus, lymph nodes, and spleen.

Human T cell subpopulations (Tμ and Tγ) were examined for their distribution in the peripheral blood, cord blood, bone marrow, tonsils, thymus, lymph nodes and spleen. The proportions of Tμ and Tγ cells are comparable in the peripheral blood, tonsils and bone marrow. The proportions of Tγ cells in cord blood are significantly higher than those in the peripheral blood. Almost complete lack of Tγ cells was observed in lymph nodes. Spleen has very high proportions of Tγ cells. Thymuses have very low proportions of both Tμ and Tγ cells when compared with peripheral blood, cord blood, tonsils, and bone marrow. The receptors for IgM on Tμ cells appear to be masked by passively absorbed IgM and require prior in vitro incubation in medium containing fetal calf serum for the full expression of this marker.