The lymphocyte surface. II. Separation of Fc receptor, C'3 receptor and surface immunoglobulin-bearing lymphocytes

A one-step separation procedure is described for both depleting and obtaining in pure form Fc receptor (FcRL), C'3 receptor (CRL) and surface immunoglobulin bearing (IgL) lymphocytes from rat lymphoid populations. The method is a modification of the Bӧyum (1968) technique for separating lymphocytes from whole blood by sedimentation on Ficoll/Isopaque, and is based on the fact that when a lymphocyte forms a rosette with sensitized erythrocytes it will sediment with the red cells rather than float with the non-rosetting lymphocytes. The technique is > 99.5% efficient at depleting thoracic duct lymphocytes (TDL) of FcRL, CRL and IgL and these subpopulations can be recovered 93-98% pure. The total recovery of lymphocytes applied is usually > 90% and the separated lymphocytes are > 95% viable. This technique allowed the cellular distribution of Fc receptors, C'3 receptors and surface Ig to be determined. It was found that ( a ) Almost all CRL carry surface Ig, although a very small sub-population of CRL (0.2-0.8%) which lacks surface Ig could regularly be detected. ( b ) A substantial proportion of IgL (12-25%) lacks C'3 receptors. ( c ) IgL and CRL which lack Fc receptors are more frequent in spleen and lymph nodes than in TDL. The proportion of this subpopulation increases in TDL after prolonged thoracic duct drainage. ( d ) Some FcRL exist which lack both C'3 receptors and surface Ig. These cells are more evident in TDL after prolonged thoracic duct drainage and in lymph nodes (20-30% of FcRL) than in early TDL or spleen (5-10% of FcRL). ( e ) The thymus contains very few FcRL, CRL or IgL. ( f ) A large population of lymphocytes exists in B rats (32-42% of TDL) which is killed by an anti-B serum but which lacks surface Ig. These cells are much less frequent in normal TDL ( < 5%) and probably also lack Fc and C'3 receptors. ( g ) Large lymphocytes probably shed their Fc and C'3 receptors, but retain their surface Ig, during S-phase. ( h ) Studies on a secondary anti-DNP response showed that a substantial proportion of direct and indirect plaque forming cells (PFC) in the spleen express Fc receptors, whereas only indirect PFC carry C'3 receptors. Virtually all PFC ( > 98%) possess surface Ig.

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THE ROLE OF LYMPHOCYTES IN THE SENSITIZATION OF RATS TO RENAL HOMOGRAFTS

Journal of Experimental Medicine ◽

10.1084/jem.122.2.347 ◽

1965 ◽

Vol 122 (2) ◽

pp. 347-360 ◽

Cited By ~ 78

Author(s):

S. Strober ◽

J. L. Gowans

Keyword(s):

Lymph Nodes ◽

Thoracic Duct ◽

Femoral Vein ◽

F1 Hybrid ◽

Duct Cells ◽

Degree Of Sensitization ◽

Spleen And Lymph Nodes

In order to study the role of blood-borne small lymphocytes in the sensitization of rats to renal homografts 2 techniques for the perfusion of isolated rat kidneys were employed: (a) the in vitro perfusion of kidneys with thoracic duct cells suspended in either an artificial medium or in blood; the perfusates were then injected into rats syngeneic with the lymphocyte donors; (b) the in vivo perfusion of kidneys with blood issuing from the femoral artery and returning to the femoral vein of living rats. The degree of sensitization conferred on the recipients by the perfusates was assessed by applying a skin homograft from the kidney donor and scoring the epithelial necrosis at 6 days. The in vitro experiments indicated that parental strain thoracic duct cells, which had passed through an F1 hybrid kidney could confer upon a parental rat sensitivity to an F1 skin graft. Several perfusions with radioactively labelled lymphocytes showed that the injected cells migrated to the lymph nodes and spleen of the recipients Labelled large pyroninophilic cells were occasionally seen in the spleen and lymph nodes of recipients, and it was suggested that these had arisen from the injected cells. Although the in vitro perfusions with blood indicated that renal homografts might sensitize their hosts within 1 hour, the in vivo perfusions suggested that about 5 to 12 hours were required. The more rapid sensitization in vitro was possibly due to the more frequent opportunity for contact between lymphocytes and kidney vascular endothelium which was afforded by the conditions in vitro.

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Identification of rat T and B lymphocytes by surface marker analysis

Laboratory Animals ◽

10.1258/002367782780908715 ◽

1982 ◽

Vol 16 (1) ◽

pp. 20-26

Author(s):

Adelaide W. Koestner ◽

A. Koestner ◽

S. Krakowka ◽

Sue S. Ringler

Keyword(s):

Lymph Node ◽

Lymph Nodes ◽

Peripheral Blood ◽

Peripheral Blood Lymphocytes ◽

Complement Receptors ◽

Tissue Sections ◽

Surface Immunoglobulin ◽

Cellular Suspension ◽

Surface Marker Analysis ◽

Spleen And Lymph Nodes

The majority of thymocytes in suspension (72·1%) formed nonimmune rosettes with guineapig erythrocytes but not with erythrocytes from sheep, cats, dogs, pigs, or man. In contrast, only a minority of cells from lymph node (2·1%) or spleen (1·4%) rosetted with guinea pig erythrocytes. Treatment of guineapig erythrocytes with neuraminidase or 2-S-aminoethyl-isothiouronium bromide did not enhance rosette formation. Adherence of the erythrocytes to tissue sections was achieved in the thymic cortex and T-cell-dependent areas of lymph node and spleen. Absorbed equine anti-rat antithymocyte serum bound 98% thymocytes and 70·0, 35·1 and 44·6% lymphocytes from peripheral blood, spleen and lymph nodes respectively. Surface immunoglobulin was demonstrated on 2·2% thymocytes, 35% peripheral-blood lymphocytes and 43 and 51% cells from lymph nodes and spleen respectively. Complement receptors were determined in suspension with erythrocyte-antibody-complement complexes. Rosetting was observed in 44% splenic, 29% lymph node and 3% thymocyte cellular suspension. The presence of Fc receptors for IgG was assessed by determining the pattern of binding of erythrocyte-antibody complexes to frozen tissue sections. For rats, antithymocyte serum is the method of choice for T cells, whereas SIg determination is the most reliable B-cell marker.

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The class of surface immunoglobulin on cells carrying IgG memory in rat thoracic duct lymph: the size of the subpopulation mediating IgG memory.

Journal of Experimental Medicine ◽

10.1084/jem.143.5.1122 ◽

1976 ◽

Vol 143 (5) ◽

pp. 1122-1130 ◽

Cited By ~ 31

Author(s):

D W Mason

Keyword(s):

Thoracic Duct ◽

Large Population ◽

Thoracic Duct Lymph ◽

Cell Sorter ◽

Heavy Chains ◽

Surface Immunoglobulin ◽

Membrane Immunoglobulin ◽

Igg Synthesis ◽

Duct Lymph ◽

Rule Out

The fluorescence activated cell sorter was used to determine the class of immunoglobulin on the thoracic duct lymphocytes (TDL) which carried IgG memory. Although only about 3% of all TDL carried membrane IgG these cells accounted for most, if not all, of the adoptive IgG anti-DNP response. It is concluded that both CR+ and CR- cells mediating IgG memory in rat TDL bear the same class of membrane immunoglobulin as that secreted by their differentiated progeny. The class of membrane immunoglobulin on CR+ and CR- rat TDL was also examined. It was found that IgM+ cells, which made up over 80% of all Ig+ cells, were virtually all CR+. In contrast, the few percent of IgG+ and IgA+ cells present were to be found in both subpopulations. There was no evidence of a large population of B cells bearing exclusively heavy chains other than IgA, IgG, of IgM. The observation that some IgG+ cells as well as IgM+ cells possess a receptor for C3 appears to rule out the hypothesis that this receptor is involved in blocking a switch from IgM to IgG synthesis.

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Early cellular events in a systemic graft-vs.-host reaction. I. The migration of responding and nonresponding donor lymphocytes.

Journal of Experimental Medicine ◽

10.1084/jem.141.3.664 ◽

1975 ◽

Vol 141 (3) ◽

pp. 664-680 ◽

Cited By ~ 42

Author(s):

R C Atkins ◽

W L Ford

Keyword(s):

Lymph Nodes ◽

Thoracic Duct ◽

Parental Strain ◽

Reference Population ◽

Third Party ◽

Slight Reduction ◽

Radioactive Label ◽

F1 Hybrid ◽

Spleen And Lymph Nodes ◽

Donor Lymphocytes

A systemic graft-vs.-host (GVH) reaction was initiated by the intravenous injection of parental strain thoracic duct lymphocytes (TDL) into irradiated F1 hybrid recipients with in-dwelling thoracic duct cannulae. The migration of the donor lymphocytes was followed by labeling them in vitro with either [3H] or [14C]uridine and measuring radioactivity by scintillation counting of the spleen and lymph nodes of the recipients removed 24 h after injection and in TDL collected throughout this period. The localization of labeled cells was always compared to that of a reference population of nonreactive lymphocytes, e.g. F1 hybrid, labeled with the alternative isotope (Fig. 1). A consistent surplus of the reactive label was found in the spleen which was balanced by a deficit of the reactive label in TDL; lymph nodes gave intermediate values. The same distribution pattern was noted when the reference population was a specifically unresponsive population of the parental strain. This differential distribution depends on recognition of the recipient's Ag-B antigens because when normal lymphocytes were injected together with specifically unresponsive lymphocytes into a "third party" F1 hybrid (against which both populations were reactive) there was no surplus of the normal cells in the spleen and no deficit in the lymph. Moreover in an Ag-B identical strain combination there was no detectable difference in the distribution of reactive and nonreactive populations. The distribution of a labeled reaction population can be accounted for if a substantial minority of cells are immobilized in the spleen and lymph nodes as a consequence of antigen recognition (Fig. 3). When the donor cells in the spleen were assayed 24 h after injection there was paradoxically a slight reduction in their specific GVH activity, which is at least partly because they are under-represented in a single cell suspension. The size of the splenic surplus (23%) and the thoracic duct deficit (12%) suggested that the minority of nonimmune lymphocytes which recognize each Ag-B complex carry 12% of the radioactive label in the original population. It is argued that this provides a near estimate of the frequency of T lymphocytes which can recognize each Ag-B antigenic complex.

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The lymphocyte surface. I. Relation between Fc receptors, C'3 receptors and surface immunoglobulin

Proceedings of the Royal Society of London Series B Biological Sciences ◽

10.1098/rspb.1974.0060 ◽

1974 ◽

Vol 187 (1086) ◽

pp. 47-63 ◽

Cited By ~ 48

Keyword(s):

Cell Surface ◽

Thoracic Duct ◽

Fc Receptors ◽

Lymphocyte Surface ◽

Surface Immunoglobulin ◽

General Properties ◽

Antigen Antibody

Fc receptors, C'3 receptors and immunoglobulin (Ig) were detected on the surface of rat thoracic duct lymphocytes by a series of rosetting procedures. This paper describes the rosetting methods and some of the general properties of these cell surface components. It was found that the Fc receptors were blocked by antigen-antibody complexes and anti─Ag-B antibodies, they were lost in vitro at 37 °C, and they were not detected in the presence of 10 -4 M azide. In contrast, the C'3 receptors were destroyed by treatment with trypsin, were insensitive to azide, and were not blocked by antigen-antibody complexes or anti─Ag-B anti-bodies. Both receptors were detected readily at 20 or 37 °C but not at 0 °C. Neither the Fc or C'3 receptors capped with surface Ig. From these differences in behaviour it was concluded that the Fc receptors, C'3 receptors and surface Ig probably represent separate molecules on the lymphocyte surface.

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Soufeng sanjie formula alleviates collagen-induced arthritis in mice by inhibiting Th17 cell differentiation

Chinese Medicine ◽

10.1186/s13020-021-00448-9 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Di Hua ◽

Jie Yang ◽

Qinghai Meng ◽

Yuanyuan Ling ◽

Qin Wei ◽

...

Keyword(s):

Cell Differentiation ◽

Lymph Nodes ◽

Inflammatory Cytokines ◽

Th17 Cell ◽

Collagen Induced Arthritis ◽

Expression Levels ◽

Th17 Cell Differentiation ◽

Spleen And Lymph Nodes ◽

Seed Coats

Abstract Background Rheumatoid arthritis (RA) is a chronic autoimmune disease. Soufeng sanjie formula (SF), which is composed of scolopendra (dried body of Scolopendra subspinipes mutilans L. Koch), scorpion (dried body of Buthus martensii Karsch), astragali radix (dried root of Astragalus membranaceus (Fisch.) Bge), and black soybean seed coats (seed coats of Glycine max (L.) Merr), is a traditional Chinese prescription for treating RA. However, the mechanism of SF in treating RA remains unclear. This study was aim to investigate the anti-arthritic effects of SF in a collagen-induced arthritis (CIA) mouse model and explore the mechanism by which SF alleviates arthritis in CIA mice. Methods For in vivo studies, female DBA/1J mice were used to establish the CIA model, and either SF (183 or 550 mg/kg/day) or methotrexate (MTX, 920 mg/kg, twice/week) was orally administered to the mice from the day of arthritis onset. After administration for 30 days, degree of ankle joint destruction and serum levels of IgG and inflammatory cytokines were determined. The balance of Th17/Treg cells in the spleen and lymph nodes was analyzed using flow cytometry. Moreover, the expression levels of retinoic acid receptor-related orphan nuclear receptor (ROR) γt and phosphorylated STAT3 (pSTAT3, Tyr705) in the spleen were detected by immunohistochemistry. Furthermore, the effect of SF on Th17 cells differentiation in vitro was investigated in CD4+ T cells under Th17 polarization conditions. Results SF decreased the arthritis score, ameliorated paw swelling, and reduced cartilage loss in the joint of CIA mice. In addition, SF decreased the levels of bovine collagen-specific IgG in sera of CIA mice. SF decreased the levels of inflammatory cytokines (TNF-α, IL-6, and IL-17A) and increased the level of IL-10 both in the sera and the joint of CIA mice. Moreover, SF treatment rebalanced the Th17/Treg ratio in the spleen and lymph nodes of CIA mice. SF also reduced the expression levels of ROR γt and pSTAT3 (Tyr705) in the spleen of CIA mice. In vitro, SF treatment reduced Th17 cell generation and IL-17A production and inhibited the expression of RORγt, IRF4, IL-17A, and pSTAT3 (Tyr705) under Th17 polarization conditions. Conclusions Our results suggest that SF exhibits anti-arthritic effects and restores Th17/Treg homeostasis in CIA mice by inhibiting Th17 cell differentiation.

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EXTH-14. A NOVEL LONG-ACTING INTERLEUKIN-7 AGONIST, NT-I7, INCREASES CYTOTOXIC CD8 CELLS AND ENHANCES SURVIVAL IN MOUSE GLIOMA MODELS

Neuro-Oncology ◽

10.1093/neuonc/noaa215.368 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii89-ii89

Author(s):

Subhajit Ghosh ◽

Ran Yan ◽

Sukrutha Thotala ◽

Arijita Jash ◽

Anita Mahadevan ◽

...

Keyword(s):

T Cells ◽

Lymph Nodes ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Cervical Lymph Nodes ◽

Cd8 Cells ◽

Interleukin 7 ◽

Long Acting ◽

Spleen And Lymph Nodes ◽

Mouse Glioma

Abstract BACKGROUND Patients with glioblastoma (GBM) are treated with radiation (RT) and temozolomide (TMZ). These treatments can cause prolonged severe lymphopenia, which is associated with shorter survival. NT-I7 (efineptakin alfa) is a long-acting recombinant human IL-7 that supports the proliferation and survival CD4+ and CD8+ cells in both human and mice. We tested whether NT-I7 would protect T cells from treatment-induced lymphopenia and improve survival. METHODS C57BL/6 mice bearing intracranial tumors (GL261 or CT2A) were treated with RT (1.8 Gy/day x 5 days), TMZ (33 mg/kg/day x 5 days) and/or NT-17 (10 mg/kg on the final day of RT completion). We followed for survival and profiled CD3, CD8, CD4, FOXP3 in peripheral blood over time. In parallel, we assessed cervical lymph nodes, bone marrow, thymus, spleen, and the tumor 6 days after NT-I7 treatment. RESULTS Median survival in mice treated with NT-I7 combined with RT was significantly better than RT alone (GL261: 40d vs 34d, p< 0.0021; CT2A: 90d vs 40d, p< 0.0499) or NT-I7 alone (GL261: 40d vs 24d, p< 0.008; CT2A: 90d vs 32d, p< 0.0154). NT-17 with RT was just as effective as NT-I7 combined with RT and TMZ in both GL261 (40d vs 47d) and CT2A (90d vs 90d). NT-I7 treatment significantly increased the amount of CD8+ cells in the peripheral blood and tumor. NT- I7 rescued CD8+ T cells from RT induced lymphopenia in peripheral blood, spleen, and lymph nodes. NT-I7 alone or NT-I7 in combination with RT increased the CD8+ T cells in peripheral blood and tumor while reducing the FOXP3+ T-reg cells in the tumor microenvironment. CONCLUSIONS NT-I7 protects T-cells from RT induced lymphopenia, improves cytotoxic CD8+ T lymphocytes systemically and in the tumor, and improves survival. Presently, a phase I/II trial to evaluate NT-I7 in patients with high-grade gliomas is ongoing (NCT03687957).

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