Lymphocyte recognition of lymph node high endothelium. VII. Cell surface proteins involved in adhesion defined by monoclonal anti-HEBFLN (A.11) antibody

Lymphocyte entry into lymph nodes (LN) and Peyer's patches (PP) occurs specifically at high endothelial cell venules (HEV). We previously isolated a high endothelial binding factor (HEBFLN) from rat lymph that blocked the lymphocyte binding sites of HEVLN but not HEVPP. In this study, mouse monoclonal anti-HEBFLN antibody (A.11) was used to investigate rat lymphocyte surface structures mediating adhesion to high endothelium. The A.11 antigen was expressed on the majority of thoracic duct lymphocytes (TDL), spleen, LN, PP cells, but was only detected on few (1 to 10%) thymus and bone marrow cells (indirect immunofluorescence). The treatment of TDL with the A.11 IgG blocked their ability to bind to HEVLN. This effect was specific, inasmuch as A.11 antibody did not block lymphocyte binding to HEVPP, and an anti-leukocyte-common antigen monoclonal antibody, OX1, did not block lymphocyte binding to HEVLN. In addition, the A.11 antigen isolated from the lymph and detergent lysates of TDL by antibody affinity chromatography had the capacity to block the lymphocyte binding sites of HEVLN but not HEVPP. Immunoprecipitation studies revealed that the A.11 antibody recognized the radioiodinated surface membrane proteins of TDL and TDL-derived T cells and B cells, which resolved with SDS-PAGE autoradiography into three polypeptides with relative m.w. of approximately 135,000, 63,000, and 40,000. We conclude that the A.11 antigen is a component of the lymphocyte surface recognition structure that mediates adhesion to high endothelial cells of rat peripheral lymph nodes.     

Lymphocyte recognition of lymph node high endothelium. VI. Evidence of distinct structures mediating binding to high endothelial cells of lymph nodes and Peyer's patches

Lymphocytes migrate from blood into lymph nodes (LN) and Peyer's patches (PP) of rats specifically at segments of venules lined by high endothelium (HEV). We previously identified and isolated a lymphocyte surface component termed high endothelial binding factor (HEBF) that appears to be involved in lymphocyte adhesion to high endothelial cells of LN. HEBF has also been isolated from thoracic duct lymph and is antigenically related to the cell surface component. Soluble HEBF derived from detergent lysates of thoracic duct lymphocytes (TDL) or directly from lymph has affinity for HEVLN in vitro, and is able to block sites where lymphocytes would normally attach. In the present study, lymphocyte binding sites of HEVLN and HEVPP were investigated through the use of lymph-derived HEBF and rabbit antibody to this factor. The results show that treatment of rat TDL with anti-HEBF Fab did not block binding to HEVPP, even though adhesion to HEVLN was reduced by 80% or more. Similarly, HEBF isolated by anti-HEBF F(ab')2 affinity chromatography blocked lymphocyte binding sites of HEVLN but not HEVPP. This material is therefore designated HEBFLN, and antibody to it is designated anti-HEBFLN Ig. Fractionation of thoracic duct lymph revealed that it contained an antigenically distinct component, HEBFPP, which blocked lymphocyte binding to HEVPP but not to HEVLN. Lymph components precipitating between 40 and 60% (NH4)2SO4 saturation contained both factors, which were separated from the bulk of lymph proteins by DEAE-Sepharose chromatography and then from each other by fractionation on the anti-HEBFLN F(ab')2-Sepharose column. The unbound fraction from this column contained HEBFPP, which was then partially purified by CM-Sepharose filtration. HEBFPP appeared to be a glycoprotein because it was destroyed by trypsin, bound to lentil lectin, and was eluted with alpha-methyl-mannoside. Together, the results demonstrate the existence of two antigenically distinct species of HEBF, and imply that lymphocyte binding sites of HEVLN and HEVPP are structurally different. We interpret the results to mean that distinct high endothelial adhesion molecules on lymphocytes mediate their entry into LN and PP.     

Rapid anti-helminthic response of B lymphocytes in the intestinal mucosal tissues of rats

B cell response to Trichinella spiralis (Ts) adult antigen (Ag) was studied in rats 1-20 days postinfection. B cell recoveries from the mesenteric lymph node (MLN), Peyer's patches (PP), thoracic duct lymph (TDL), and the spleen were determined by FACS analysis and Ag-specific antibody-producing cells (Ab-pc) in these tissues were enumerated using the immunoplaque assay. Total B cell numbers increased 2-70 times from day 3 postinfection in the MLN and TDL obtained from MLN-resected rats (MX) and such proliferation was not found in the PP or the spleen. Ab-pc of all isotypes increased from day 3 in the MLN and from day 2 in the MX-TDL. Among all isotypes, IgE- and IgG1-pc showed the strongest response. Immunofluorescence study revealed that these B cells were activated in the non-PP region of the small intestine. These results indicate an early isotype switch to IgG1 and IgE production in Ts-infected small intestine.     

Lymphocyte adhesion to cultured Peyer's patch high endothelial venule cells is mediated by organ-specific homing receptors and can be regulated by cytokines

Adhesion of lymphocytes to high endothelial venule (HEV) cells is the first step in the migration of these cells from blood into lymph nodes and Peyer's patches (PP). In the present study, we isolated and cultured HEV cells from PP of the rat and assessed their capacity to interact with lymphocytes. Flow cytometric analysis with a rat HEV-specific mAb KJ-4 revealed that greater than 90% of the cultured cells were stained by the antibody. Furthermore, confluent monolayers of PP HEV cells retained the capacity to support the adhesion of lymphocytes from spleen, thoracic duct, and lymph nodes but not binding of immature cells from thymus and bone marrow, which are deficient in cells capable of binding to HEV in vivo. In addition, intraepithelial lymphocytes that preferentially migrated into mucosal lymphoid tissues were also enriched in cells that adhered to the endothelial monolayers. The binding process required energy, was calcium-dependent, and could be inhibited by cytochalasin D, trypsin, and mixed glycosidase. Interestingly, pretreatment of PP HEV cells with rTNF, IFN-gamma, or granulocyte-macrophage CSF significantly increased the endothelial adhesiveness for thoracic duct lymphocytes in a time- and dose-dependent manner. In contrast, stimulation of lymphocytes with phorbol ester or TNF resulted in the rapid modulation of the surface expression of the PP homing receptor and decrease in lymphocyte binding to normal or TNF-stimulated HEV cells. The adhesion of lymphocytes to normal or cytokine-stimulated HEV cells can be blocked by pretreatment of lymphocytes, but not HEV cells, with the PP homing receptor-specific 1B.2.6 antibody. Taken together, these experiments provide strong evidence that the interaction between lymphocytes and cultured HEV cells are mediated by adhesive mechanisms that regulate lymphocyte entry into PP in vivo and that cytokines can promote HEV adhesiveness for lymphocytes through increased expression of organ-specific ligands on HEV cells.     

Transforming growth factor-beta 1 and IL-4 regulate the adhesiveness of Peyer's patch high endothelial venule cells for lymphocytes.

The adhesion of lymphocytes to endothelial cells lining the postcapillary high endothelial venules (HEV) is the first step in their emigration from the bloodstream into lymph nodes and Peyer's patches (PP). We have recently shown that the adhesiveness of cultured rat lymph node and PP HEV cells for thoracic duct lymphocytes can be increased significantly by pretreatment with TNF-alpha, IFN-gamma, and IL-4. In the present study we investigated the role of transforming growth factor-beta 1 (TGF-beta) on the adhesiveness of nonstimulated and cytokine-stimulated PP HEV cells for rat lymphocytes. The results indicated that at picomolar concentrations, TGF-beta significantly (p less than 0.001) decreased the ability of PP HEV cells to adhere 51Cr-labeled rat lymphocytes. Maximal inhibition was observed with a TGF-beta dose of 0.5 ng/ml and an incubation time of 6 to 12 h. TGF-beta did not affect the morphology of HEV cells and had no adverse effect on their viability. Moreover, the decrease in HEV adhesiveness by TGF-beta was reversible, with lymphocyte binding returning to control level 24 h after removal of the cytokine. The specificity of TGF-beta was confirmed by the ability of neutralizing anti-TGF-beta 1 antibody, but not control serum, to abolish the inhibitory properties of the cytokine. In addition, TGF-beta completely abrogated the increased adhesiveness of PP HEV cells normally induced by TNF-alpha or IFN-gamma. In contrast, TGF-beta had no effect on the stimulating effects of IL-4. Moreover, preincubation of PP HEV cells with TGF-beta did not alter the ability of these cells to respond to IL-4. Importantly, the adhesion of rat lymphocytes to IL-4-stimulated PP HEV cells can be blocked by pretreatment of lymphocytes with the PP-homing receptor-specific 1B.2.6 antibody whereas pretreatment of human mononuclear cells with anti-very late activation antigen-4 alpha antibody inhibited only partially the binding of these cells to the IL-4-stimulated PP HEV monolayers. Taken together, these findings strongly suggest that TGF-beta and IL-4 play important regulatory roles in lymphocyte-HEV adhesion and that the stimulatory effect of IL-4 is mediated at least in part through the increased expression of organ-specific ligands on HEV cells.     

Characterization of recirculating lymphocytes in rheumatoid arthritis patients: selective deficiency of natural killer cells in thoracic duct lymph

Five patients with rheumatoid arthritis (RA), who were treated by lymphocyte depletion by using thoracic duct drainage (TDD), provided an opportunity to characterize the phenotype and function of their recirculating lymphocytes. We found that: a) thoracic duct lymphocytes (TDL) were similar in their proportion of T cells (83% +/- 6 OKT3+), OKT4+ subset (65% +/- 8), and OKT8+ subset (22% +/- 6) to peripheral blood lymphocytes (PBL): b) fewer natural killer-like cells were present in TDL (5% +/- 4 Leu-7+; 2% +/- 2 Leu-11+: 8% +/- 2 OKM -1+) than in PBL (20% +/- 10 Leu-7+: 11% +/- 6 Leu-11+; 18% +/- 5 OKM -1) (p less than 0.01); c) TDL differed from synovial fluid lymphocytes ( SFL ) and synovial membrane lymphocytes ( SML ) in that TDL lacked a high percentage of activated lymphocytes (T cells bearing Ia antigen, OKT10 , and transferrin receptor): d) immature T cells (expressing either OKT6 antigen or reactive with peanut agglutinin) were not found in TDL even late in the course of TDD: and e) in vitro functional studies demonstrated that TDL were similar to PBL in their ability to synthesize immunoglobulin after mitogen stimulation and to generate cytotoxic T lymphocytes capable of lysing autologous EBV-transformed B cells. However, natural killer activity, as measured by lysis of K562 cells was significantly lower in TDL than PBL (p less than 0.05). These results demonstrate that natural killer cells defined by phenotype and function are excluded from thoracic duct lymph and thus have a circulation pattern different from most T cells.     

A monoclonal antibody specific for rat intestinal lymphocytes

A monoclonal antibody, RGL-1, was produced by fusion of NSI myeloma cells with spleen cells of a mouse immunized with isolated rat intraepithelial lymphocytes (IEL). SDS-PAGE analysis revealed that RGL-1 precipitated two major noncovalently bound chains of about m.w. 100,000 and 125,000, and a minor component of m.w. 200,000. Examination of both tissue sections and isolated cells indicated that RGL-1 stained the majority of the lamina propria lymphocytes and IEL but only very few cells (less than 2%) in the lymphoid organs and small numbers of lymphocytes in other mucosae. In the small intestine, RGL-1 stained lymphocytes with the helper (W3/25) as well as the cytotoxic/suppressor (OX8) phenotype. The antibody reacted with 95% of the granular IEL but with less than 0.1% of the blood large granular lymphocytes. Although mature IgA plasma cells in the lamina propria were RGL-1-, some large IgA-containing cells were weakly positive. In the gut-associated lymphoid tissues (GALT), studies combining immunofluorescence and autoradiography indicated that 56 and 73% of rapidly dividing cells of mesenteric lymph nodes and of thoracic duct lymph (TDL) stained with RGL-1, respectively. In addition, 90 to 100% of the IgA-containing blasts of MLN and 75% of those of TDL were labeled by RGL-1. In contrast, rapidly dividing cells of spleen and of peripheral lymph nodes did not stain with RGL-1. Because RGL-1 can be demonstrated on both intestinal lymphocytes and their immediate precursors in the GALT, its expression may be related to the homing of lymphocytes into the gut mucosa.     

An in vitro model of lymphocyte homing. II. Membrane and cytoplasmic events involved in lymphocyte adherence to specialized high-endothelial venules of lymph nodes.

Rat thoracic duct lymphocytes (TDL) are capable of selective adherence to the endothelium of high-endothelial venules (HEV) when overlaid onto glutaraldehyde-fixed sections of lymph nodes. The data presented indicate that lymphocyte adherence is an energy-dependent, calcium-requiring event that involves membrane determinants on TDL which are sensitive to trypsin. Surface sialic acids on lymphocytes are not essential and treatment of the cells with neuraminidase does not interfere with their attachment to HEV. There was no evidence that microtubule-associated functions play a role in binding. Adherence, however, is abolished by cytochalasin B, indicating that the cytoplasmic contractile microfilament system exerts an important effect. The results imply that lymphocyte surface membrane modulation is involved in the development of strong adhesive forces that bind the cells to the endothelium. In addition, lymphocyte-HEV adherence is reduced by ionophore A-23187, an agent known to inhibit surface membrane receptor movement. It is suggested that specific binding of recirculating lymphocytes to HEV is not a passive event, but that activation of cytoplasmic contractile forces in the lymphocyte is required for the formation of stable lymphocyte-HEV binding.     

Inhibition of lymphocyte endothelial adhesion and in vivo lymphocyte migration to cutaneous inflammation by TA-3, a new monoclonal antibody to rat LFA-1.

Lymphocyte function-associated Ag-1 (LFA-1) or CD11a/CD18 mediates lymphocyte adhesion to cultured vascular endothelial cells (EC). Thus, LFA-1 likely plays a major role in lymphocyte migration out of the blood, but there is little information on this in vivo. Small peritoneal exudate lymphocytes (sPEL) and lymph node (LN) lymphoblasts adhere to cytokine-activated EC and preferentially migrate to cutaneous inflammatory sites. The role of LFA-1 in the adherence and in vivo migration of these T cells was determined. Because of a lack of anti-rat LFA-1, mAb were prepared to rat T cells. One mAb, TA-3, inhibited homotypic aggregation; T cell proliferation to Ag, alloantigens, and mitogens; stained all leukocytes; and immunoprecipitated 170- and 95-kDa polypeptides from lymphocytes and neutrophils. TA-3 binding to lymphocytes also required Ca2+, but not Mg2+. Thus, TA-3 appears to react with rat LFA-1. TA-3 inhibited spleen T cell adhesion to unstimulated EC by 30% and to IFN-gamma, TNF-alpha, IL-1 alpha, and LPS stimulated EC by 50 to 60% but inhibited sPEL EC adhesion by only 10%. TA-3 also strongly inhibited anti-CD3-stimulated LN T cell adherence. The migration of spleen T cells to delayed-type hypersensitivity and skin sites injected with LPS, poly I:C, IFN-gamma, IFN-alpha/beta, and TNF was inhibited by 72 to 88% by TA-3, and was decreased by 50% to peripheral LN. TA-3 caused less but still 50 to 60% inhibition of sPEL migration to inflamed skin. Lymphoblast migration to skin was inhibited 40 to 80% and to PLN by 30%. Migration of lymphocytes from all sources to mesenteric LN was inhibited by 32 to 60%. In conclusion, LFA-1 mediates much of the adherence of spleen T cells and lymphoblasts to EC in vitro, most of the migration of these cells to dermal inflammation and about 50% of the homing of LN and spleen T cells to peripheral and mesenteric LN. sPEL are less dependent on LFA-1 for adhesion to EC in vitro and for migration to inflamed skin and LN in vivo.     

Vitamin B6 deficiency and the lymphoid system. II. Effects of vitamin B6 deficiency in utero on the immunological competence of the offspring.

Results of this study indicated that an absence of vitamin B₆ from the diet of pregnant rats led to reduced immunological competence in the offspring. While the numbers of cells in the thoracic duct lymph (TDL) of rats approximately 3 months old and progeny of vitamin B₆-deficient mothers were nearly equivalent to control values, such cells had a reduced capacity to respond in the mixed lymphocyte and normal lymphocyte transfer reactions. It is suggested that this reduction may have reflected (i) an alteration in the capacity of TDL cells or their precursors to give rise to immunologically competent cells, (ii) a shift in the proportions of T and B cells in the TDL, and/or (iii) an absence or ineffectiveness of a humoral factor required for the development of immunologically competent lymphocytes.     

The migration of lymphocytes across specialized vascular endothelium. VI. The migratory behaviour of thoracic duct lymphocytes retransferred from the lymph nodes, spleen, blood, or lymph of a primary recipient

Thoracic duct lymphocytes labelled with 51Cr were injected into a primary recipient and then were transferred for a second time from the lymph nodes (cervical and/or mesenteric), spleen, lymph, or blood into a series of final recipients. Measurement of the organ distribution of labelled lymphocytes in the final recipients enabled three main conclusions to be drawn. (1) Lymphocytes that had localized in the spleen, mesenteric lymph nodes (LN), or cervical LN of the first recipient showed no tendency to return in increased numbers to the same organ in the final recipient. (2) Lymphocytes that had recently entered the spleen or LN were temporarily impaired in their ability to reenter LN. This capacity was recharged when the cells returned to the lymph and the blood. (3) Lymphocytes that had been passaged from blood to lymph and collected for up to 4 hr at room temperature entered the LN of a recipient much faster than did nonpassaged thoracic duct lymphocytes collected overnight at 0 degree C. Supplementary experiments indicated that the different migratory behavior of thoracic duct lymphocytes under these two circumstances was mainly a consequence of their handling in vitro during the collecting and the labelling procedures. This functional impairment was not associated with a diminished ability to enter the spleen and bone marrow or to survive in recipients for up to 24 hr.     

Biochemical identification of rat ART-1 and Ly-1 alloantigens

The results of this study indicate that the ART-1 and Ly-1 rat alloantigens are synonymous with each other and also with the leukocyte-common (L-C) antigen which has been previously identified as a major glycoprotein of rat thymocytes and T and B lymphocytes. This conclusion is supported by the following observations: (i) when labeling of rat lymphoid cells was studied with a fluorescence-activated cell sorter, the profiles obtained were similar for labeling with ART-1 and Ly-1 alloantibodies and a monoclonal antibody to L-C antigen: (ii) this labeling was almost completely inhibited by purified L-C antigen: (iii) preincubation with L-C antigen completely inhibited binding of the alloantibodies in a cellular radioimmunoassay; (iv) the cytotoxic effect of the alloantibodies was completely abolished by preincubation with purified L-C antigen; (v) the strain distribution of the ART-1 and Ly-1 alloantigens was identical for 11 rat strains and in linkage analysis the ART-1 and Ly-1 alloantigens were found to cosegregate.Genetic linkage studies have shown that the L-C antigen locus is unlinked to the major histocompatibility antigen (RT1), the immunoglobulin light chain (1^k) and to the coat color gene (C) loci.Abbreviations used in this paper BSA Bovine serum albumin - DAB Dulbecco's salt solution - FCS Fetal calf serum - L-C antigen Leucocyte-common antigen - LN Lymph node - TDL Thoracic duct lymphocytes     

Negative selection experiments support the idea that T-T help is required for the H-Y-specific cytotoxic T cell response

Antigen-primed female thoracic duct lymphocyte (TDL) populations (B6, H-2K^bI-A^bD^b) were depleted of T cells specific for the male H-Y antigen presented in the context of I-A^b, by negative selection through irradiated male H-2K^bI-A^bD^b recipients. This ocedure substantially removed the capacity of such TDL to respond to H-Y-H-2D^b. The response can be reconstituted by adding either smaller numbers of unfiltered, H-Y-primed, B6 female TDL or anti-Ly-2-treated memory T cells. The results thus support the idea that concurrent presence of helper T cells specific for H-Y-H-2-I-A^b is required for the generation of secondary cytotoxic T lymphocyte responses to H-Y-H-2D^b. This is discussed in the context of similar, though not identical, experiments with the vaccinia virus model.     

Antigenic relationship between medullary thymocytes and a subpopulation of peripheral T cells in the rat: description of a masked antigen.

Using differentially absorbed rabbit antisera to rat thoracic duct cells, an antigen is described which normally is expressed on the surface of T cells in thoracic duct lymph and lymph node, but which exists in a masked form on medullary thymocytes and apparently not at all on cortical thymocytes. This antigen is termed the rat masked thymocyte antigen (RMTA). RMTA on medullary thymocytes can be unmasked mechanically by sectioning in a cryostat or enzymatically by treating with neuraminidase. Trypsin destroys or removes RMTA. Nearly all the T cells in thoracic duct lymph and lymph node are RMTA⁺, whereas only 58–66% of T cells in spleen are RMTA⁺. RMTA⁺ T cells, which are cortisone resistant, reside in the paracortex and periarteriolar sheath regions of lymph node and spleen. RMTA^? T cells, which are cortisone sensitive, appear to reside in the red pulp of spleen. The results suggest that (i) two antigenically distinct populations of T cells exist in the rat, RMTA⁺ and RMTA^? T cells, (ii) medullary thymocytes are the immediate precursors of RMTA⁺ T cells, and (iii) cortical thymocytes may be the immediate precursors of RMTA^? cells.     

Influenza A virus interaction with murine lymphocytes. I. The influence of influenza virus A/Japan 305 (H2N2) on the pattern of migration of recirculating lymphocytes.

The effect of influenza virus A/Japan 305 (H2N2) on the path of migration of recirculating lymphocytes has been studied. 51Cr-labeled rat thoracic duct lymphocytes (TDL) were incubated with virus at 37 degrees C for 1 hr and then infused i.v. into syngeneic recipients which were killed 1 hr later. Virus-treated TDL accumulated in the liver and their recovery in lymph nodes and spleen was severely reduced. Changes in lymphocytes induced by virus developed rapidly and were evident after incubation for only 15 min. UV-irradiated virus altered the pattern of lymphocyte localization but attachment of heat-inactivated virus to lymphocytes in vitro had no effect on their distribution in vivo. Evidence was obtained that some virus-treated TDL, initially sequestered in the liver, subsequently recovered their ability to circulate normally. Recovery was not complete and a population of cells failed to regain their ability to home into lymph nodes. Evidence is also presented demonstrating that influenza virus affected the homing properties of both T and B cells. It is suggested that aberrations in lymphocyte homing were mediated by the viral neuraminidase which induces changes in the cell membrane leading to their accumulation in the liver.     

The circulating life span, immunocompetence and 3H-uridine uptake of small lymphocytes from thymus-grafted, neonatally thymectomized rats.

By 7 weeks post-grafting, the number of small lymphocytes in the thoracic duct lymph (TDL) and blood of the thymus-grafted neonatally thymectomized adult rats had increased to 60% of the number of cells in sham controls, or 2-1/2 times thymectomized control values. This increasing consisted almost exclusively of long-lived, recirculating small lymphocytes and corresponded to a 60% recovery of cellular immunocompetence as measured by the mixed lymphocyte reaction (MLR). Associated with the return of cellular immunocompetence was an increased incorporation of 3H-uridine by the small lymphocytes. Cells from thymectomized animals grafted with lymph node fragments demonstrated no significant increase in lymphocyte numbers nor was there a return of immunocompetence as compared to thymectomized controls.     

Interaction of thymus lymphocytes with histoincompatible cells. IV. Mixed lymphocyte reactions of activated thymus lymphocytes

CS7BL-activated CBA T cells (T.TDL) were obtained by thoracic duct cannulation of (CBA × C57BL)F₁ mice 4 days after heavy irradiation and injection of CBA thymus cells. T.TDL behaved differently from the TDL of normal CBA mice in unidirectional mixed lymphocyte culture in a number of respects: (a) the response of T.TDL was directed specifically against C57BL antigens, whereas normal TDL responded to both C57BL and BALB/c antigens; (b) the response of T.TDL was rapid but transient compared to that of TDL; (c) whereas only approximately 3% of TDL synthesized DNA specifically in response to C57BL antigens, as many as 25% of C57BL-activated T.TDL responded to these antigens. Evidence is presented which suggests that the T.TDL have a very limited capacity to proliferate. Most of the cells which responded to antigen synthesized DNA without subsequently entering mitosis.     

Migration of recently divided B and T lymphocytes to peritoneum and lung

It is recognized that a population of newly divided (or young) cells migrate preferentially to inflamed foci. It has been shown that a large proportion of lymphocytes residing in the bronchoalveolar airspaces of rat are recently divided cells and that blood may be an important source of these cells. To further delineate how blood may contribute to lymphocyte subpopulations in inflamed peritoneum and lung, a comparison of the capacity of recently divided T and B cells to migrate from blood to inflamed peritoneum and lung was made. To label young lymphocytes, DA strain donor rats were given Initiated thymidine by vein in vivo for 7 days. After thoracic duct drainage, the following labeled cell populations were adoptively transferred by vein into syngeneic recipients: (i) unseparated thoracic duct lymphocytes (TDL), (ii) enriched T cells (>90%) or B cells (>80%) recovered after passage of TDL through nylon columns, and (iii) thoracic duct lymphocytes (> 99% B cells) obtained from “B rats” that were prepared by X irradiation, thymectomy, and bone marrow reconstitution. T and B cells were identified by specific heterologous antisera. The percentage recovery of labeled lymphocytes in the recipients with inflamed peritoneum or lung aspirates was determined from cell counts and autoradiographs. The studies indicated that (a) both labeled T and B cells migrated to inflamed peritoneum and lung; (b) labeled B cells migrated to peritoneum and lung better than did labeled TDL or T cells; and (c) labeled lymphocytes did not migrate to unstimulated peritoneum. The enhanced migration of newly divided B lymphocytes to inflamed peritoneum and normal lung (a site that is likely under chronic antigenic stimulation) was unexpected, but may provide additional information on the relative contribution of these subpopulations in the immune inflammatory response.     

Migration patterns of dendritic cells in the rat: comparison of the effects of gamma and UV-B irradiation on the migration of dendritic cells and Lymphocytes

To further define the underlying mechanisms of immune suppression induced by UV-B irradiation, we have examined the kinetics of homing patterns of in vitro UV-B-irradiated and gamma-irradiated-thoracic duct lymphocytes (TDL) compared to dendritic cells (DC). Our findings show that 111In-oxine-labeled TDL specifically home to the spleen, liver, lymph nodes, and bone marrow with subsequent recirculation of a large number of cells from the spleen to lymph nodes. In contrast, DC preferentially migrate to the spleen and liver with a relatively insignificant distribution to lymph nodes and an absence of subsequent recirculation. Splenectomy prior to cell injection significantly diverts the spleen-seeking DC to the liver but not to the lymph nodes, while the homing of TDL to lymph nodes is significantly increased. In vitro exposure of 111In-oxine labeled TDL to gamma irradiation does not significantly impair immediate homing to lymphoid tissues but inhibits cell recirculation between 3 and 24 hr. In contrast, gamma irradiation does not affect the tissue distribution of labeled DC, suggesting that DC are more radioresistant to gamma irradiation than TDL. Unlike the findings in animals injected with gamma-irradiated cells, UV-B irradiation virtually abolished the homing of TDL to lymph nodes and significantly reduced the homing of the spleen-seeking DC to the splenic compartment while a large number of cells were sequestered in the liver. The results of in vitro cell binding assay show that TDL, unlike DC, have the capacity to bind to high endothelial venules (HEV) within lymph node frozen sections while gamma and UV-B irradiation significantly inhibit the binding of TDL to lymph node HEV. These findings suggest that: (i) DC, unlike TDL, are unable to recirculate from blood to lymph nodes through HEV; (ii) although gamma irradiation impairs TDL recirculation, it does not affect DC tissue distribution; and (iii) UV-B irradiation impairs both TDL and DC migration patterns. We conclude that the lack of capacity of irradiated TDL to home to lymph nodes is due to damage to cell surface homing receptors and that the failure of DC to home to the lymph node microenvironment is related to the absence of HEV homing receptors on their cell surface.     

Transfusion induces blood donor-specific suppressor cells

Transfusion with blood from the organ donor before transplantation can prolong the survival of renal allografts in the rat. To determine if the beneficial effect of preoperative blood transfusion was due to the generation of donor-specific suppressor cells, in vivo and in vitro adoptive transfer experiments were performed. Lymphoid cells were harvested from transfused and untreated rats. These cells were then either (1) transferred to lightly irradiated (200 R) syngeneic hosts which were subsequently challenged with a kidney allograft (in vivo assay) or (2) titrated as regulator cells into naive unidirectional MLC such that the regulator and responder populations were syngeneic. In the LEW-RT1 to DA-RT1av1 strain combination, the adoptive transfer of thoracic duct lymph (TDL) or lymph node (LN) cells (5 x 10(7) to 7.5 x 10(7) cells) from DA animals transfused with LEW blood, 7 days previously into syngeneic (DA), lightly irradiated (200 R) hosts resulted in the indefinite survival of LEW kidney allografts. The phenomenon was blood donor-specific and dose-dependent. In contrast the adoptive transfer of spleen cells (10(7) to 10(8] from blood transfused hosts 7 days after transfusion had no effect on renal allograft survival. In vitro the addition of LN or TDL regulator cells, harvested from DA rats transfused with LEW blood, to a unidirectional MLC (DA responders, LEW stimulators) resulted in a significant depression of the proliferative response when compared with the proliferation of these same cells without the addition of these regulator cells or with the addition of LN or TDL regulator cells from a DA rat transfused with third party (PVG-RT1c) blood. The depression of the proliferative response observed in vitro, was blood donor specific. When LN or TDL regulator cells from a DA rat transfused with PVG-RT1c blood were added to a unidirectional MLC between DA responders and PVG stimulators, a significant depression in the proliferative response was observed. These in vitro findings were confirmed in two other strain combinations (LEW-PVG, and DA-PVG). Thus a single blood transfusion results in the induction of donor-specific suppressor cells detectable both in vivo and in vitro 7 days after transfusion in some but not all lymphoid compartments.