The migration of lymphocytes across specialized vascular endothelium. IV. Prednisolone acts at several points on the recirculation pathways of lymphocytes

Radioactively labelled thoracic duct lymphocytes from syngeneic rat donors were injected iv into recipients which had been given a continuous iv infusion of prednisolone at 1 mg/hr for 15–18 hr previously. The tissue distribution and recirculation into lymph of the labelled lymphocytes were compared quantitatively in the prednisolone-treated and control recipients by scintillation counting and autoradiography. The most prominent effect of prednisolone was to retard recirculating lymphocytes within the tissues to which they are normally distributed by the blood, namely the bone marrow, the spleen, and the lymph nodes. Although lymphocyte traffic was almost completely frozen by prednisolone, recirculating lymphocytes were not killed. A second effect of prednisolone was to impair the influx of lymphocytes from the blood into lymph nodes. Different groups of lymph nodes varied in the extent to which prednisolone inhibited the entry of lymphocytes, and previous antigenic stimulation completely exempted lymph nodes from this inhibition. Lymphocytes took a longer time to cross the walls of high endothelial venules in the lymph nodes of prednisolone-treated rats. A third effect of prednisolone was to increase the rate at which lymphocytes entered the bone marrow from the blood by crossing sinusoidal endothelium.     

THE TEMPO OF LYMPHOCYTE RECIRCULATION FROM BLOOD TO LYMPH IN THE RAT

Radioactively labelled thoracic duct lymphocytes were obtained either by incubation in vitro with ³H-uridine or ¹⁴C-uridine or by giving potential donors repeated injections of ³H-thymidine finishing 17 days before thoracic duct cannulation. These labelled TDL were injected i.v. into syngeneic recipients which had been subjected to splenectomy and thoracic duct cannulation on the previous day. The tempo of lymphocyte recirculation from blood to lymph was reflected by the time at which radioactivity was recovered in the thoracic duct lymphocyte output of the recipient. This was measured by scintillation counting of 2-hourly fractional collections for 36 hr after the injection. Two lines of evidence showed that the majority of small lymphocytes which label intensely with radioactive uridine in vitro were uniform in their 'migration potential'with a modal blood to lymph transit time of 14–18 hr. By contrast the cells which were labelled in vivo with ³H-thymidine included a slower population with a modal transit time of 24–28 hr. These conclusions can be more fully interpreted in the light of recent evidence on thymic-independent ('B') lymphocytes.     

The influence of strong transplantation antigens (Ag-B) on lymphocyte migration in vivo.

The tissue localization of syngeneic thoracic duct lymphocytes was compared to that of allogeneic cells in four rat strain combinations differing at the Ag-B locus (HO → DA, DA → HO, AO → HO, HO → AO). Dual isotope labeling with [³H]uridine and [¹⁴C]uridine was applied in order so that the distribution of allogeneic and syngeneic cells could be followed in one recipient. During the first couple of hours after iv injection, allogeneic lymphocytes usually migrated as easily into the various tissues as did syngeneic cells. However, after 24 and 48 hr, a reduced amount of label associated with allogeneic cells was often measured in the tissues. This reduction differed in magnitude in the different strain combinations and was most pronounced in the lymph nodes. A reduced number of allogeneic cells also appeared in the thoracic duct. By contrast, no reduced localization of allogeneic lymphocytes was measured in the draining popliteal lymph nodes late after sc injection. In preimmunized animals allogeneic cells were rapidly removed from the blood and therefore failed to localize in the lymphoid tissues. Furthermore, the lymph node localization of allogeneic cells was more like that of syngeneic cells in splenectomized rats, as well as in irradiated recipients (when the irradiation was given shortly before cell transfer). It is concluded that transplantation antigens play no essential role in the interaction between recirculating lymphocytes and the venous endothelium at the sites where the large-scale physiological emigration of the cells takes place (the HEVS of the lymph nodes and the marginal zone vessels of the spleen). The elimination of allogeneic cells is found later; it probably takes place in the lymph nodes and spleen. Possible mechanisms responsible for this rapid removal of allogeneic lymphocytes in nonimmunized recipients are discussed.     

The entry of T and B lymphocytes into rat popliteal lymph nodes undergoing a graft-versus-host reaction

The entry of radiolabeled blood-borne T and B lymphocytes into resting popliteal lymph nodes and popliteal lymph nodes stimulated with semiallogeneic lymphocytes was investigated in rats. Thoracic duct lymphocytes separated into T- and B-lymphocyte populations on nylon-wool columns were radiolabeled with 51chromium and equal numbers of T or B lymphocytes were injected intravenously. While the ratio of T and B lymphocytes in the blood is approximately 3:1 it was found that the ratio of T to B lymphocytes migrating into lymph nodes was approximately 9 T to 1 B lymphocyte in both resting and antigenically stimulated lymph nodes. Since the ratio of T to B lymphocytes in thoracic duct lymph is similar to that of blood, there is a disparity between the number of T cells entering and leaving lymph nodes. These results suggest that some T lymphocytes may return to the blood directly and/or there is increased T lymphocyte death in lymph nodes.     

Freezing of rat lymphocytes. 2. Distribution and circulation of frozen-thawed 51Cr-labelled cells

Radioactive labelled synegenic fresh and frozen-thawed rat lymphoid cells were injected into groups of animals and their distribution in liver, spleen, lung, lymph nodes, and thoracic duct recorded. Principally the same pattern of distribution was demonstrated after injection of both fresh and frozen-thawed cells. However, more radioactivity was recovered from the liver and less from lymph nodes and thoracic ducts in recipients of frozen-thawed than of fresh cell preparations. This is caused by some damage to the cells in the freeze-thaw process.     

Comparative migration of B- and T-Lymphocytes in the rat spleen and lymph nodes.

B-lymphocytes were obtained either by thoracic duct cannulation of thymectomized, irradiated rats or by isolation of complement-receptor-bearing lymphocytes from normal rats. They were labeled in vitro with [³H]-leucine and injected iv into syngeneic recipients from which samples of spleen and lymph node were taken at intervals from 15 min to 48 hr after injection. The sites of initial localisation of B- and T-lymphocytes were identical suggesting that the cells migrated into both organs by a common entrance. The two cell types remained closely associated for several hours in the paracortex of lymph nodes and at the periphery of the periarteriolar lymphoid sheath of the spleen. After 1–6 hr, B-cells segregated from T-cells by moving on into the adjacent part of the lymphocyte corona in the follicular area. By 24 hr, B-cells were evenly distributed throughout the corona. A definite minority of B-cells but no T-cells were seen within the germinal centres. In the spleen, T-cells moved into the central area of the periarteriolar sheath before returning to the blood. The immunological significance of the routes of B- and T-cell migration is discussed.     

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.     

Natural killer cell activity in the rat. V. The circulation patterns and tissue localization of peripheral blood large granular lymphocytes (LGL)

Large granular lymphocytes (LGL) and T cells were separated from blood by centrifugation on discontinuous gradients of Percoll, were labeled with [3H]uridine or [111In]oxine, and were injected i.v. into syngeneic euthymic or athymic nude rats. The tissue distribution of these labeled cells was monitored for up to 24 hr after transfer by scintillation counting of tissue homogenates and autoradiography of tissue sections. In normal euthymic rats, the main sites of LGL localization were the alveolar walls of the lungs and spleen red pulp; however, they were not detectable in the major traffic areas of T lymphocyte recirculation, the spleen white pulp, and lymph nodes. Furthermore, the density of labeled LGL was very low in the small intestine, thymus, kidney, and liver, although on a per-organ basis, about 10% of the injected radioactivity was found in the liver by 24 hr post-injection. When 111In-labeled LGL were injected i.v. into rats with an indwelling thoracic duct cannula, they completely failed to enter the thoracic duct lymphocyte (TDL) population over an observation period of 6 days. This finding was markedly different from the results obtained with T cells and was consistent with the lack of natural killer and antibody-dependent cellular cytotoxicity activity observed among TDL, even in rats pretreated with the biological response modifier, poly I:C. LGL in athymic nude rats also failed to recirculate between blood and lymph. However, in contrast to normal euthymic animals, a significant increase in the localization of radiolabeled LGL to lymph nodes was observed in nude rats between 30 min and 24 hr. Taken as a whole, these findings define the areas within the lungs and spleen in which blood LGL normally localize, and clearly demonstrate that LGL do not normally recirculate between blood and lymph.     

The migration of lymphocytes across specialized vascular endothelium. II. The contrasting consequences of treating lymphocytes with tryspin or neuraminidase.

Lymphocytes were exposed in vitro to either trypsin or neuraminidase. The ability of the treated cells to migrate into tissues were measured (a) by i.v. injection into intact recipients and (b) by vascular perfusion through an isolated lymph-node preparation. The localization of trypsinized cells in the lymph-nodes of recipients was deficient when compared to untreated lymphocytes and there was a surplus of trypsinized cells in blood. Trypsinized cells migrated into the isolated nodes in reduced numbers. By contrast, neuraminidase treated lymphocytes were markedly deficient in the blood of recipients early after injection; their localization in the spleen and lymph-nodes was also deficient but they were in surplus in the liver. Moreover they migrated into the isolated nodes in slightly increased numbers. By 24 hr after injection the perturbed localization pattern produced by either enzyme was partly restored to normal. In conclusion, tryspin interfered with the capacity of lymphocytes to migrate into lymph-nodes but neuraminidase did not; the latter promoted the hepatic sequestration of cells and the reduced localization in the blood and tissues was a consequence of this. The hypothesis that lymphocytes adhere to specialized endothelia in lymph-nodes because of specific glycoside sequences on their surface lacks experimental support.     

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.     

STUDIES ON LYMPHOCYTES

Continuous extracorporeal irradiation of the circulating blood (ECIB) of from 3 to 501/2 hr duration was used to study in the calf the differential depletion of lymphocytes from spleen, lymph nodes and thymus as compared to blood and thoracic duct lymph. The cell content of tissues was measured by planimetry and/or test point analysis. Lymphocyte depletion by ECIB from various lymphoreticular organs, and from different areas within a given organ, was less than in the circulating blood or the thoracic duct lymph and varied from one site of a lymphoreticular organ to another. The degree of depletion with time followed an exponential function with at least two components. The first component corresponded to a relatively rapid fall and the second to a very slow reduction in lymphocyte content. The former is related to the elimination of an easily mobilizable pool of lymphocytes while the latter corresponds to a more sessile mass of lymphocytes which exchange with blood lymphocytes very slowly. Elimination of the easily mobilizable pool of lymphocytes by ECIB from all tissues studied was observed within 10–15 hr, indicating that the rate of exchange with blood is similar for this group of cells in various lymphoreticular tissues. The size, however, of the easily mobilizable vs the more sessile pools of lymphocytes may vary considerably, the best estimates for the former being as follows (in per cent of total lymphocyte mass): lymph node medulla, less than 10%; lymph node cortex plus paracortical zone, 18% (depletion mainly paracortical); red pulp of the spleen, 37%; densely populated white pulp of the spleen, 55%; and loosely populated white pulp of the spleen, 60%. In comparison, the approximate fractions of lymphocytes originating fromthe easily mobilizable pools in various lymphoreticular tissues plus the cells already circulating a t the onset of EClB correspond to 64% for the thoracic duct lymph and 78% for the circulating blood respectively. These findings are discussed in relation to production, recirculation and life span of lymphocytes, and immune reactions.     

Random recirculation of small T lymphocytes from thoracic duct lymph in the mouse

The migration of ⁵¹Cr-labeled nylon-wool separated mouse thoracic duct T cells has been followed in order to determine whether there is a circulation of small (nondividing) T cells through the small intestine. Approximately 6% of the injected dose of T-TDL localized in the small intestine (minus Peyer's patches). Experiments revealed that this gut-localizing cell population consisted almost entirely, if not exclusively, of lymphoblasts present in mouse T-TDL. When lymphoblasts and small lymphocytes from mouse T-TDL were separated by velocity sedimentation, and the migration of separated fractions was studied, we found large cells (66% blasts) migrated well to the gut but poorly to the lymph nodes, whereas small cells (2% blasts) showed minimal migration to the gut but localized randomly in lymph nodes and spleen. The in vivo distribution of small cells from T-TDL was similar to that of T-PLN. Furthermore, the recirculatory patterns of both ⁵¹Cr-labeled T-TDL and T-PLN were found to be identical as accessed by their rate of recovery in the thoracic duct lymph of recipient mice. These results support the notion that the vast majority of T-TDL and T-PLN are part of a common pool of recirculating T cells which recirculate randomly through lymph nodes and spleen and not the small intestine.     

Circulating T and B lymphocytes of the mouse. I. Migratory properties

Studies on the identity of thoracic duct lymphocytes (TDL⁴) from normal and T cell-depleted mice indicated that as many circulating B lymphocytes were produced by healthy T cell-depleted mice as normal mice. Proportions of T and B cells from the thoracic duct of CBA mice changed markedly during the first 4 days of drainage from 82% T cells and 16% B cells at 12 hr to approximately equal proportions of both classes after 3 days. In absolute terms, T cells were mobilized rapidly by thoracic duct drainage and B cells very slowly. Histologically, this was reflected in a rapid depletion of the T cell-dependent areas of the lymphoid organs. The B cell-dependent areas, in contrast, became depleted of lymphocytes only after drainage for a week or more.The homing properties of circulating lymphocytes were investigated using TDL from normal and T cell-depleted mice as relatively pure sources of T and B cells, respectively. Four hours after injection of ⁵¹Cr-labeled T and B cells, a large proportion of both cell classes were found in the spleen. By 24 hr, many T cells had left the spleen and appeared in the lymph nodes. Such redistribution by B cells, however, was minimal.Intravenously injected T and B cells, labeled with tritiated uridine (³HU), localized specifically in the T and B cell-dependent areas, respectively, of the lymphoid tissues.³HU-labeled T cells were found to recirculate rapidly from blood to lymph. Labeled B cells, in contrast, recirculated only very slowly.     

Migration pathways of recirculating murine B cells and CD4+ and CD8+ T lymphocytes

Migration pathways of B cell and CD4+ and CD8+ T cell subsets of murine thoracic duct lymphocytes (TDL) were mapped. Per weight, the spleen accumulated more TDL than any other organ, regardless of lymphocyte subset. Spleen autoradiographs showed early accumulations of TDL in marginal zone and red pulp. Many TDL exited the red pulp within 1 hr via splenic veins. The remaining TDL entered the white pulp, not directly from the adjacent marginal zone but via distal periarterial lymphatic sheaths (dPALS). From dPALS, T cells migrated proximally along the central artery into proximal sheaths (pPALS) and exited the white pulp via deep lymphatic vessels. B cells left dPALS to enter lymphatic nodules (NOD), then also exited via deep lymphatics. T cells homed to lymph nodes more efficiently than B cells. Lymphocytes entered nodes via high-endothelial venules (HEV). CD4+ TDL reached higher absolute concentrations in diffuse cortex than did CD8+ T cells. However, CD8+ TDL moved more quickly through diffuse cortex than did CD4+ TDL. B cells migrated from HEV into NOD. Both T and B TDL exited via cortical and medullary sinuses and efferent lymphatics. A migration pathway across medullary cords is described. All TDL subsets homed equally well to Peyer's patches. T TDL migrated from HEV into paranodular zones while B cells moved from HEV into NOD. All TDL exited via lymphatics. Few TDL entered zones beneath dome epithelium. All subsets were observed within indentations in presumptive M cells of the dome epithelium.     

The stable and permanent expansion of functional T lymphocytes in athymic nude rats after a single injection of mature T cells

Athymic nude rats (PVG.rnu/rnu) were injected at 6 to 10 wk of age with 1 to 200 million thoracic duct lymphocytes (TDL) containing 40 to 60% mature T cells. Thereafter TDL-injected nude recipients were monitored for evidence of T cell function for up to 2 yr. W3/25+ T helper (Th) cells in lymph nodes (LN) increased from 7% at 2 wk to 30% at 8 wk after TDL transfer. The percent of W3/25+ cells remained elevated for the life of the recipient (up to 2 yr), approximating normal levels. The total size of the recirculating pool expanded in TDL-injected nude rats to reach 2/3 the level of euthymic controls by 16 wk, an increase of 10-fold to 15-fold in W3/25+ cells. The expansion of the W3/25+ population was independent of initial TDL dose. With time spleen and LN acquired a normal histological appearance including the development of germinal centres and a marked increase in cellularity in T cell traffic areas. TDL-injected nude rats rejected skin allografts with near normal kinetics. In addition graft vs host (GVH) responsiveness, assessed by the popliteal LN assay, progressively increased reaching a level 9 mo to 1 yr after replacement that resembled the GVH activity in euthymic controls.     

Adrenergic innervation of lymph nodes and the thoracic duct

By means of incubation of slices in 2% solution of glyoxylic acid distribution of adrenergic fibers in the rabbit lymph nodes and in the thoracic lymphatic duct has been studied. Adrenergic fibers get into parenchyma of the lymph nodes via two ways. The first--the perivascular, when the nervous fibers make a plexus and get into the node along the blood vessels, the second--diffuse nervous fibers get together with trabecules in between the lymphoid nodules. The distribution density of the adrenergic fibers is not the same in different groups of the lymph nodes. In the lumbar nodes it is the highest. In the lymph nodes of the cervical part the density of the sympathetic fibers is, as a rule, lower than in the lumbar, but higher than in the axillary nodes. The lowest density of th adrenergic fibers is in the mesenteric, superficial inguinal lymph nodes and in the lymph nodes, situating near the thoracic part of the aorta. In the lymphatic duct wall small amount of adrenergic fibers are revealed, they form a plexus, predominantly in the cranial part.     

Recruitment of effector lymphocytes by initiator lymphocytes. Circulating lymphocytes are trapped in the reacting lymph node.

In previous studies, we have shown that initiator T lymphocytes (ITL) sensitized in vitro recruit effector T lymphocytes in vivo. When ITL were sensitized against fibroblast antigens in vitro and injected into footpads of syngeneic recipients, they induced enlargement of the draining popliteal lymph node (PLN) and the development there of specific effector lymphocytes of recipient origin. To study the basis of this lymph node response in recruitment, we injected 51Cr-labeled spleen cells i.v. into recipients of sensitized ITL and found that the labeled circulating lymphocytes were trapped in the reacting PLN. The trapping depended on surface properties of the labeled circulating lymphocytes, as revealed by various enzymatic treatments. The trapping process was radiosensitive, both on the part of the trapped lymphocytes and the lymph node-trapping mechanism. Thus, sensitized ITL injected into the hind footpads migrate to the PLN and induce the trapping of circulating recruitable lymphocytes, which either differentiate into or regulate the differentiation of effector T lymphocytes.     

Allelic allotype inclusion and exclusion among rabbit Ig-bearing lymphocytes of peripheral blood and lymphoid organs

Mononuclear cells isolated from peripheral blood, appendix, sacculus rotundus, mesenteric lymph nodes, and spleen of b⁴b⁵ heterozygous rabbits were examined for surface Ig allotypes of the b locus. Ig allotype-bearing cells were detected as cells binding erythrocytes or bacteria coated with monospecific anti-b4 or anti-b5 antibody (Ab). Rosetting the cells with Ab-coated erythrocytes indicated that many peripheral blood lymphocytes, but relatively few appendix cells, bore both the b4 and b5 allotypes. Lymphocytes bearing both the b4 and b5 allotypes were also detected by incubating the cells with a mixture of Escherichia coli coated with anti-b4 Ab and Gaffkya tetragena coated with anti-b5 Ab. The percentage of Ig-positive lymphocytes binding both bacteria was 22–31% in the peripheral blood, 4–6% in the appendix, 3–5% in the sacculus rotundus, 4–10% in the mesenteric lymph nodes, and 5% in the spleen. Thus, the percentage of double-bearing lymphocytes was higher in the blood than in the appendix, sacculus rotundus, mesenteric lymph nodes, or spleen. The b4b5-bearing cells in the blood were not cells with adsorbed cytophilic Ab, since these cells still bore both the b4 and b5 allotypes after pronase digestion and Ig regeneration. These double-bearing lymphocytes, i.e., cells exhibiting allelic allotype inclusion, are probably less differentiated cells.     

Characterization of cellular and molecular immune effectors against Trichinella spiralis newborn larvae in vivo.

The cellular and molecular immune effectors that participated in host immunity against Trichinella spiralis newborn larvae were characterized in vivo using AO rats. Donor rats were immunized with 2,000 muscle larvae orally or 11,400 newborn larvae i.v. Immune serum and cells from spleen, peripheral lymph nodes, mesenteric lymph node, thoracic duct lymph and the peritoneal cavity were obtained from donor rats 10-21 days after infection and transferred into normal recipient rats. The control recipients received either no cells and serum or normal cells and normal serum obtained from normal donors. Newborn larvae (20,000-50,000) were injected either i.v. or ip into these recipients and immunity against newborn larvae was measured either by muscle larvae burden of the recipients three weeks later or by direct recovery of newborn larvae from the peritoneal cavity of the recipients. The experiments demonstrated that immune lymphocytes conferred no protection in the recipients but that immune serum and immune peritoneal cells were protective and these effects were synergistic. Cell adherence to the cuticle and killing of newborn larvae were observed in the peritoneal cavity of immune rats. Positive fluorescence was observed on newborn larvae incubated with fractionated IgM and IgG(E) antibody isotypes. Massive deposition of antibody molecules on newborn larvae was demonstrated by scanning electron microscopy. Studies using transmission electron microscopy revealed that the larval adherent cells were stimulated macrophages, neutrophils and eosinophils.     

Nonspecific entry of thoracic duct immunoblasts into intradermal foci of antigens

Lymph borne immunoblasts were obtained by collecting thoracic duct lymph from inbred rats 3–5 days after either killed C. parvum, B. abortus or B.C.G. organisms had been injected subcutaneously into the hindquarter regions to stimulate the caudal lymph nodes. By incubating the lymph cells with a radioactive precursor of DNA, 5-iodo-2-deoxyuridine-¹²⁵I, the immunoblasts became labelled but the small lymphocytes did not. The labelled cells were washed and injected intravenously into syngeneic recipients which had had intradermal injections of various antigens at various previous times. The entry of labelled cells into these injection sites was monitored by counting the radioactivity that they contained up to 24 hr later.It was found that the accumulation of radioactivity in the skin lesions was maximal 12 hr after the donor cells had been injected, but the immunological specificity of the donor immunoblasts did not affect significantly the extent to which they entered lesions which contained the same or unrelated antigens. It was found also that the sites of intradermal injections of B.C.G. or C. parvum always attracted more immunoblasts than sites containing other antigens; this was a non-specific effect, thought to be related to the adjuvant properties of these organisms.