Fate of Autogenous Canine Lymphocytes after Injection into an Afferent Lymphatic of the Popliteal Lymph Node

Dog thoracic duct lymphocytes were labeled in vitro with ³H-uridine and infused into an afferent lymphatic of the popliteal lymph node of the same dog. Ten minutes after infusion nearly all the injected radioactivity was recovered from the lymph node. An effect of infusion flow rate on the percentage of cells retained by the lymph node was observed ½ to 3½ hours after infusion, and was probably mediated by the tendency of the node to become edematous after infusions at a rate exceeding 0.045 ml/min. Edematous nodes retained 83.7% of the cells, as compared to 47.5% for nonedematous nodes. As early as 30 minutes after infusion a small amount of ³H-radioactivity was found in the spleen and thoracic duct lymph. The deep iliac and paraaortic nodes on the side of the infusion contained significant amounts of ³H-radioactivity, while negligible amounts were detected in the contralateral popliteal node at any time. The intranodal localization of the ³H-labeled cells was studied by radioautography. All labeled cells remained intrasinusoidal during the first 4 hours after infusion. At 9 and 21 hours some labeled cells were located in the extrasinusoidal parenchymal lymphoid tissue of the cortex and the medulla, but the majority still remained intrasinusoidal.     

Lymph node localization and whole body distribution of radioiodinated encephalitogenic polypeptide in guinea-pigs

A bovine encephalitogenic polypeptide (BEP) labelled with radioiodide retained its capacity to induce experimental encephalomyelitis (EAE). Guinea-pigs were injected with ¹²⁵I BEP in Freund's complete adjuvant (FCA), to study changes in the architecture and the distribution of radioactivity in draining lymph nodes, and the amount of radioactivity in various organs.

After injection of BEP in FCA the lymph node rapidly enlarged. Within 48 hr there was depletion of lymphocytes, the enlarging lymphoid follicles had become confluent and there was proliferation of large `epithelioid' cells throughout the node. At 5 days the lymph node architecture was disorganized and lymph follicles with germinal centres could not be recognized; similar but less pronounced changes were present in regional nodes. By contrast, after injection of flagellin in FCA, there were numerous lymphocytes, plasmablasts and pyroninophilic cells, germinal centres were prominent, and the architecture was preserved.

From 0·5 to 0·8% of the total injected radioactivity was concentrated in the popliteal lymph node 2–5 days after injection of ¹²⁵I BEP in FCA. No radioactivity was concentrated in the node after injection of ¹²⁵I BEP without FCA, and animals thus immunized did not develop encephalomyelitis.

The popliteal lymph node was examined by autoradiography after injection of ¹²⁵I BEP in FCA. At 24 hr radioactive encephalitogen associated with droplets of adjuvant was present mainly in the peripheral sinus and at 48 hr encephalitogen–adjuvant droplets were deposited randomly throughout cortex and medulla. These droplets appeared to represent sites where lymphoid cells acquired their capacity for pathogenic reactivity with their target antigen in the central nervous system.

     

Effect of blockage of the afferent lymphatic vessels on the development of popliteal lymph nodes in the rat

The effect of blockage of the afferent lymphatic vessels on the development of popliteal lymph nodes in the rat was studied. The afferent lymphatic vessels to each popliteal node were surgically interrupted at the lowest edge of the popliteal fossa at 3, 7 or 28 days after birth and the popliteal nodes were obtained from treated animals at 4, 8 or 16 weeks after the operation. At 7 days after birth, each popliteal node was small and weighed 0.2 mg. Its parenchyma consisted of reticular cells and a small number of dispersed lymphoid elements. Four weeks after birth, each node weighed 4-5 mg, and its parenchyma comprised two layers, an outer continuous layer of peripheral cortex containing 40-50 lymph follicles and an inner discontinuous layer of deep cortex made up of 4-5 deep cortical units. At 10-12 weeks after birth, each node weighed about 10 mg and showed full structural development; the peripheral cortex contained 100-130 lymph follicles and the deep cortex consisted of 4-6 well developed units. The popliteal node was drained by 4-6 afferent lymphatic vessels, which opened into the subcapsular sinus of the node. Each lymphatic opening was topographically associated with a respective deep cortical unit, as previously described by Bélisle and Sainte-Marie (1982). In animals treated at 3 or 7 days after birth, the development of the popliteal nodes was considerably inhibited. Four weeks after surgery, each node showed 10-30 lymph follicles in the peripheral cortex and 1-3 small units in the deep cortex. Sixteen weeks after surgery, the node weighed about 4 mg and its cortex exhibited about 50 lymph follicles in the peripheral cortex and only 2 units in the deep cortex. The popliteal nodes of the treated animals generally received 2 afferent lymphatic vessels. In animals treated at 4 weeks after birth, the popliteal nodes showed no gain in weight for following 16 weeks. Four weeks after surgery, each node usually had 4-5 deep cortical units and 50-60 lymph follicles. Thereafter, some units and their overlaying peripheral cortex underwent atrophy, while others persisted. Sixteen weeks after surgery, the popliteal node showed only 2 deep cortical units and 50-60 lymph follicles, and was drained by 2 afferent lymphatic vessels. Surgical interruption of the afferent lymphatic vessels to the popliteal node at the lowest edge of the popliteal fossa did not obliterate all the draining lymphatic vessels , but reduced the number of vessels opening into the node.(ABSTRACT TRUNCATED AT 400 WORDS)     

Binding of Candida albicans yeast cells to mouse popliteal lymph node tissue is mediated by macrophages.

We previously reported that Candida albicans yeast cells adhere to the macrophage-rich medullary and subcapsular sinus areas of mouse lymph node tissue. To determine whether the yeast cell-lymph node interaction is mediated by macrophages, the effect of specific elimination of macrophages on yeast cell binding was studied, and yeast cell adherence was correlated with the ingestion of India ink by lymph node cells. Macrophage elimination was done by use of liposome-containing dichloromethylene diphosphonate (L-Cl2MDP). Mice were injected in the hind footpads with the L-Cl2MDP preparation, popliteal lymph nodes were removed 5 days later, and yeast cell adherence was determined by an ex vivo binding assay. As controls, lymph nodes from mice that received footpad injections of either phosphate-buffered saline (PBS) alone or liposome-containing PBS were used. Use of macrophage- and neutrophil-specific monoclonal antibodies in tissue immunostaining showed that the L-Cl2MDP treatment eliminated macrophages but not neutrophils from the medullary and subcapsular sinus areas of the popliteal lymph nodes. A striking reduction of yeast cell adherence occurred with lymph nodes from L-Cl2MDP-treated mice compared with lymph nodes from control animals. The lymph node-yeast cell binding patterns of L-Cl2MDP-treated and control mice were the same regardless of mouse strain, sex, or T-cell competency. Results of India ink experiments, in which India ink was injected into footpads of mice and was rapidly taken up by popliteal lymph node macrophages, showed a strong correlation between yeast adherence and India ink staining of cells. In addition, the interaction of yeast cells with lymph node tissue from normal mice was not significantly affected by the addition of two extracellular matrix proteins, fibronectin and laminin, during the ex vivo adherence assay. These data indicate that medullary and subcapsular sinus lymph node macrophages express an adhesion system similar to that described for mouse splenic marginal zone macrophages.     

Transport of immune complexes from the subcapsular sinus into the lymph node follicles of the rat

To study the mode of transport of immune complexes from the subcapsular sinus into the follicles of draining popliteal lymph nodes, horse radish peroxidase (HRP)-anti HRP was injected in rat footpads. Within six minutes, complexes were already present in the subcapsular sinus freely or attached to the plasma membrane of different types of cell including cells forming the stroma. A few minutes later, complexes were also seen in the deeper part of the outer cortex, and after two hours they had reached the periphery of the follicles. They were always seen scattered between lymphoid and non-lymphoid cells. After one day, complexes were present on well-developed follicular dendritic cells. After injection of HRP, no localization of this antigen was observed in the deeper part of the outer cortex including the follicles. These results strongly suggest that HRP-anti HRP complexes are passively transported through the outer cortex into the follicles where they are trapped and retained by follicular dendritic cells.     

A histological and experimental study on the fate of an increased number of lymph follicles produced in the mouse popliteal lymph node by exogenous antigen stimulation

Eight-week-old female C57B1/6 mice were injected with endotoxin LPS and/or other antigens into the left hind footpad, and then the number of lymph follicles in the draining popliteal lymph nodes was examined. In untreated mice each popliteal node contained 10-12 lymph follicles at both 8 and 15 weeks of age. Animals given 50 micrograms of LPS at 8 weeks of age showed an increase in the number of lymph follicles 3 weeks later, but this number returned to normal levels by 15 weeks after the LPS injection. After a 2-micrograms-LPS injection at 23 weeks of age, the number of lymph follicles in the draining lymph node was unchanged, but that in animals given the 2-micrograms-injection 15 weeks after the 50-micrograms-LPS injection was significantly increased. In animals receiving 2 Lf of diphtheria toxoid, instead of the 2-micrograms-LPS, at 15 weeks after a 50-micrograms-LPS injection, the number of lymph follicles per draining node was within the normal range. In one group of mice, the initial injection of 50-micrograms-LPS at 8 weeks of age was followed by injections every third week of several kinds of antigens which had been shown to be ineffective in inducing follicle formation. Here, the number of lymph follicles in the draining popliteal node was kept to significantly increased levels at 25 weeks of age. The present results suggest that, while most lymph follicles normally developing in the lymph node are maintained for a long time under normal conditions, many lymph follicles induced by antigenic challenge have a limited life span and undergo atrophy unless they are periodically activated by additional antigenic stimuli, and that atrophied follicles finally become unable to respond to antigenic stimulation. It is also suggested that antigenic materials which trigger the formation of lymph follicles in the primary challenge can evoke follicle formation more efficiently in the secondary challenge.     

Antigen localization in lymphopenic states: II. Further studies on whole body X-irradiation

The gross and microscopic distribution of ¹²⁵I polymerized flagellin from Salmonella adelaide was studied in adult rats at various times following 800 r whole body X-irradiation. Injections of radioactive antigen were made in both hind footpads, and the popliteal lymph nodes were excised for autoradiographic study 1 day later. This dose of irradiation caused a progressive decline in the ability of lymphoid follicles of popliteal nodes to capture and retain antigen. Irradiation had no detectable effect upon antigen uptake by whole lymph nodes or upon the number of grains overlying the phagocytic cells of the medullary sinuses of popliteal nodes.

Various substances capable of restoring follicular antigen uptake in the irradiated rat were studied by means of injecting the test substance into one hind footpad 1 hour prior to the injection of antigen into both feet. The distribution of antigen in each popliteal node was compared, each animal thus acting as its own control. It was found that 0.01 ml of specific anti-flagellar immune serum, or 0.25 ml of normal adult rat serum significantly improved follicular antigen uptake when tested ten days after irradiation. Foetal calf serum, homologous lymphocytes, and the media from pooled concentrated lymphocyte cultures were without demonstrable effects when given by regional injection. Shielding of the popliteal nodes at the time of irradiation improved follicular antigen uptake, whereas shielding of the femoral bone marrow and appendix was ineffective. No agent found capable of improving follicular antigen capture in the irradiated rat significantly altered footpad retention of antigen, whole organ counts of the popliteal nodes, or antigen localization in the phagocytic cells of the lymph node medulla.

The results favour the interpretation that the follicular antigen trapping mechanism is extremely sensitive to changes in levels of opsonins; that substances present in normal adult rat serum act as `follicular opsonins'; that these substances decline exponentially following irradiation; and that these substances are secreted by small lymphocytes or their progeny.

     

Macrophages and the activity of high endothelial venules. The effect of interferon-gamma

Interruption of the afferent lymphatic vessels of rat popliteal lymph nodes led to the disappearance of the monoclonal antibody ED3-reactive subcapsular sinus macrophages within 3 weeks, but had no effect on the ED1+ macrophages in the paracortical area. This disconnection of the afferent lymph flow to the popliteal lymph node also reduced the capacity of high endothelial venules (HEV) to bind lymphocytes and led to a flattening of the HEV. Activating factors or cells in the incoming lymph might be responsible for the maintenance and function of several different cell populations and we therefore wished to determine if the effects of interruption could be restored by injection of recombinant rat interferon-gamma (rIFN-gamma). Injection of rIFN-gamma directly into operated lymph nodes could mediate an apparent increase of ED1+ cells within 24 h but rIFN-gamma could not restore the macrophage subpopulation in the subcapsular sinus, as recognized by monoclonal antibody ED3. Restoration of the decreased binding capacity of HEV could not be observed with the doses and time points tested, suggesting that HEV are a distinct type of endothelium.     

In Vivo Behavior of Fibrinogen Fragment D in Experimental Renal, Hepatic and Reticuloendothelial Dysfunction

Fibrinogen Fg-D, obtained by plasmin-induced cleavage of fibrinogen, was separated and purified by ion exchange chromatography. The in vivo behavior was studied by administering 2 mg of ¹²⁵I-labeled Fg-D intravenously into each of 3 normal, 3 partially hepatectomized, 3 reticuloendothelial system (RES) blockaded, 4 nephrectomized and 2 ureter ligated rabbits. The plasma clearance in normal rabbits showed two components: 66.0 ± 6.0% was cleared with a t½ of 0.9 ± 0.2 hours and 32.3 ± 5.3% cleared with a t½ of 3.6 ± 0.3 hours. In both the partially hepatectomized and RES-blockaded groups, the clearance patterns were similar to those observed in the normal animals. In contrast, in the nephrectomized group, while the t½ of the first component was similar to that in the normal group, the second component had a longer t½ (6.0 ± 1.0 hours) than the other groups. In the animals with both ureters occluded, the t½'s were similar to the normal animals. Measurements of urinary radioactivity suggested that complete in vivo catabolism had occurred. In vivo subfragments of Fg-D could not be detected in the plasma. Neither Fg-D nor subfragments were found in the urine. These results indicate that Fg-D is rapidly cleared from the plasma, that in vivo subfragmentation does not occur, and that the kidneys are important in the catabolism of a portion of Fg-D.     

Antigen in tissues: IV. The effect of antibody on the retention and localization of antigen in rat lymph nodes*

A study has been made of the role of specific antibody in causing increased retention and specific localization of a weak antigen, human serum albumin (HSA), in the popliteal and aortic lymph nodes of rats. The antigen was labelled with ¹²⁵I prior to mixing with antibody. HSA mixed with excess homologous antibody was trapped to the greatest extent in these nodes after footpad injection of the antigen. Injection of HSA with antibody caused increased uptake of HSA into the medulla but retention was poor as autoradiographs showed the area to be essentially free of antigen 4–5 days after injection. By contrast, antigen injected with antibody localized strongly in lymphoid follicles and persisted at this site. Both IgM and IgG antibody were found to cause follicular localization of HSA in rats. Heterologous, isologous and homologous antibody also caused follicular localization of the antigen. Purified homologous γ-globulin was found to localize in the follicles. A moderate increase in the net negative charge of the γ-globulin by acetylation did not appreciably affect the ability of the globulin to localize in the follicles. Detectable formation of antibody did not occur in the rats after injection of antigen—antibody complexes, owing possibly to the inhibitory effect of free antibody on the primary response.     

Immunogenicity of human serum albumin: decay in the normal mouse

The disappearance of immunogenicity of human serum albumin (HSA) was measured by injection of 1 μg of this protein in normal mice and by transferring to them, at intervals, a fixed number of HSA-sensitized syngeneic spleen cells: subsequently the antigen binding capacity of the serum was determined. This system served to reveal the residual amount of immunogen present in the animal, since under these conditions the adoptive secondary response is proportional to the HSA available, and no antibody is formed by the host. The half-life of HSA immunogenicity was 17½ hours. A similar rate of decline (T = 16½ hours) was found by following ¹³¹I coupled to the HSA in the circulation of the same mice. This demonstrates that the albumin antigen is not stored in an active form in non-pre-immunized mice.     

Antibody isotypes mediating antigen retention in passively immunized mice.

Antibody isotypes vary in their capacity to mediate retention of a readily catabolized protein antigen, human serum albumin (HSA) in spleen, popliteal lymph node (PLN) and hind foot. Hyperimmune anti-HSA mouse sera were separated into fractions highly enriched for IgM, IgG1 and IgG2 via differential elution from protein A-Sepharose. These fractions were used to immunize normal mice passively. Twenty-four hours later the mice were injected with radio-iodinated HSA into the hind footpad. When the amount of HSA retained in the spleen 6 days later was determined, the potency of various antibody fractions to mediate retention could be ranked IgG2=IgG1 > IgM. The amount of HSA retention mediated by various fractions correlated well with autoradiographic evidence demonstrating localization of HSA in splenic follicles. The localization pattern in PLN was similar to the spleen except that the IgM-containing fraction mediated follicular localization of HSA to a considerable degree. In tendons of the hind foot, IgG1 mediated HSA retention five times better than IgG2 or IgM fractions. The amount of radioactivity found in the liver varied inversely with HSA retention in other locations. The results demonstrate differences in antibody isotype requirements for antigen localization in spleen, regional lymph node and collagenous sites of the hind foot.     

The histogenesis of lymph nodes in rat and rabbit

The histogenesis of the popliteal lymph node in the rat and the popliteal and inguinal lymph nodes in the rabbit was examined by light microscopy. Special emphasis has been laid on the initial lymphocyte population in the lymph node anlage. In the rat on the seventeenth day of gestation lymphoid cells populate a limited mesenchymal area along the vein wall. The next day the mesenchyme shows a bulb-shaped outgrowth causing an indentation in the wall of a lymph vessel, running parallel to the vein and having a saccular widening at this place. The bulb-shaped lymphoid outgrowth fills up the widened lymph vessel; the subcapsular sinus originates from the remaining parts of the lymph vessel. At birth the lymph node can be divided into a primitive cortex consisting of an area with evenly scattered lymphocytes among the basic network of reticular cells and a medulla. About three days after birth an ovoid area containing a dense concentration of lymphocytes is observed in the inner cortex. In the next days it expands in both lateral and medullary direction but not into the outer cortex. Primary follicles appear in the outer cortex 18 days after birth. The development of the inguinal and popliteal lymph nodes in the rabbit shows the same characteristics as the histogenesis of the popliteal lymph node in the rat. The morphogenesis of the lymph node is summarized in a schematic diagram.     

Lymph flow and lymph protein concentration in the skin and muscle of the rabbit hind limb

1. Three lymphatic beds have been found in the rabbit hind limb:

(i) the lymph from the foot and ankle drains into lymphatics which run with the deep veins to the popliteal node;

(ii) the superficial lymphatics of the medial skin from mid-calf to the groin enter the inguinal node while those of the lateral skin drain into the popliteal node;

(iii) the lymph draining the muscles collects in vessels which do not enter the popliteal node but join the femoral lymphatic post-nodally.

2. The lymphatic system of the hind limb is regionalized so that lymph from a specific area enters the popliteal node in one specific lobe and no other.

3. By cannulating the femoral lymphatic and ligating the post-nodal lymph vessel close to the point at which it leaves the node it was possible to collect pure muscle lymph.

4. The mean muscle lymph flow was 21 μl./100 g.min whilst the skin lymph flow was 240 μl./100 g.min. The mean protein concentration of muscle lymph was usually somewhat higher than that of skin lymph.

5. After nerve stimulation there was an increase in muscle lymph flow but no increase in protein concentration.

6. After a mild thermal injury there was no change in muscle lymph flow or its protein concentration, but there was an enormous increase in the leakage of lactic dehydrogenase indicating considerable cellular injury. On the other hand a significant increase in both protein concentration and flow of skin lymph occurred after thermal injury.

     

Tissue localization and retention of antigen in relation to the immune response

Antigen persists for months or even years in lymphoid tissues of immune animals and this antigen is believed to participate in the induction and maintenance of B-cell memory as well as in the maintenance of serum antibody levels. In the present report we describe the phenomenon of antigen localization and long-term retention on mouse follicular dendritic cells (FDCs). The antigens used were injected in the hind footpads of immune mice and the popliteal lymph nodes were the lymphoid organs generally studied. In addition to presenting the morphological features of mouse FDCs, we report the results of a study of the mechanism of antigen migration from the site of initial localization in the lymph node subcapsular sinus to the regions of follicular retention in the cortex. The migration was followed by light and electron microscopy. The results support the concepts that immune complexes are trapped in the subcapsular sinus and are transported by a group of nonphagocytic cells to follicular regions. The mechanism of transport may involve either migration of pre-FDCs with a concomitant maturation into FDCs, or cell-to-cell transport utilizing dendritic cell processes and membrane fluidity; or a combination of the two mechanisms may be in operation.     

The localization of non-microbial antigens in the draining lymph nodes of tolerant, normal and primed rabbits

Soluble haemocyanin (HCy) or human serum albumin (HSA) labelled with ¹²⁵I at 8 and 0·7 μc/μg, respectively, were injected into the footpads of rabbits in doses just sufficient to elicit a primary response in normal animals, and the distribution of radioactivity in the popliteal lymph nodes between 6 hours and 21 days later was studied by autoradiography. The recipient rabbits had either been primed by a single prior injection of unlabelled antigen, or made putatively tolerant by repeated neonatal administration of antigen, or were previously untreated. Localization of antigen in germinal centres, in a typical dendritic pattern, was marked in the primed animals throughout the period of observation; in those tolerant animals which had no detectable serum antibody initially and made no detectable antibody response such localization was not seen at any time; in the animals that had no previous contact with antigen there was no localization in germinal centres until about the time when antibody became detectable in the serum. Localization of radioactivity in medullary sinus macrophages did not differ significantly between the three groups.

It is concluded that localization of these soluble antigens on the dendritic web in lymphoid follicles occurs as a consequence of the presence of circulating antibody. Uptake of the antigens by medullary macrophages, however, can occur in the absence of antibody. Although the degree of labelling of medullary macrophages was not evidently affected by the presence of antibody in these experiments, it is emphasized that the antibody levels, even in the primed animals, were low, and that this finding is unlikely to apply when the amount of antibody present is relatively much greater than the amount of antigen injected.

     

The relationship between the morphology of rosette-forming cells and their mode of rosette formation

Rats were injected in the footpad with sheep erythrocytes. The proportion of each morphological type of rosette-forming cell in the popliteal lymph node was measured, in vitro, performing the immunocyto-adherence technique at 4° and 37°. Among the different morphological types of rosette-forming cell, which appear after immunization, few plasmacytes form rosettes at 4°.

The number of rosettes formed at 37° was increased by the addition of anti-rat immunoglobulin (Ig) to aliquots of lymph node preparations from rats 7 days after immunization. This increase was accounted for by an increase in the number of rosette-forming plasmacytes and rosette-forming cells intermediate between small blasts and plasmacytes, and was thought to be due to the detection of cells secreting non-haemagglutinating antibody. The enhancing effect of anti-rat Ig at 37° contrasted with its inhibitory effect at 4°. The presence of 10^-3 M puromycin only slightly reduced the number of rosettes as compared with untreated control aliquots, but it prevented or reduced the enhancing effect of anti-rat Ig. The addition of anti-rat Ig to aliquots of lymph node preparations from rats 4 days after immunization reduced the number of rosettes formed as compared with control aliquots. It is considered that plasmacytes lack a surface associated antigen-binding receptor and depend for their rosette-forming ability on the secretion of antibody.

     

Kinetics of the proliferative response to antigen in vitro of rabbit lymph node cells taken at various times after immunization

The antigen stimulation of thymidine uptake by immune rabbit lymph node cells in vitro was studied. The kinetics of the response varied depending on the time between immunization and culture. Cultures set up early after immunization showed a peak response over day 1–2 of culture while those set up late after immunization showed a peak response over day 3–4.

Studies using the metabolic inhibitor BUDR suggested that this is due at least in part to a larger recruitment of cells into the response during the third day of culture when lymph node cells taken late after immunization were used.

Removal of antigen from cultures after brief exposure of cells at 4° reduced the magnitude but did not eliminate the proliferative response, suggesting that some antigen is specifically bound to the cells in the cold. Readdition of antigen restored normal reactivity.

Holding cells at 4° for 4 hours without antigen had no effect on their response to subsequent addition of antigen. However, if cells were held at 4° for 3 hours with antigen present a severe degree of depression of subsequent thymidine incorporation was observed in some but not all experiments. This depression of responsiveness was interpreted as an in vitro phenomenon comparable to immunologic tolerance.

     

The growth of the cell population in the draining popliteal lymph node of rats injected with rat adrenal in Freund's adjuvant

The growth of the cell population and the rate of cell proliferation in the draining popliteal lymph node was studied in rats injected in the foot with adrenal tissue antigen-adjuvant emulsion and pertussis vaccine. The number of cells in the lymph node quadrupled during the first two days after injection prior to any significant increase in the rate of cell proliferation as measured by the number of cells incorporating ³H-thymidine in vitro. After the second day proliferation of cells contributed to the growth of the lymph node's cell population. It is suggested that the early sudden growth of the cell population is brought about partly by an increase in the influx of lymphocytes from the blood into the lymph node and partly by a decrease in the efflux of lymphocytes with the efferent lymph from the lymph node.     

The humoral immune response is initiated in lymph nodes by B cells that acquire soluble antigen directly in the follicles

The initial step in a humoral immune response involves the acquisition of antigens by B cells via surface immunoglobulin. Surprisingly, anatomic studies indicate that lymph-borne proteins do not have access to the follicles where naive B cells reside. Thus, it is unclear how B cells acquire antigens that drain to lymph nodes. By tracking a fluorescent antigen and a peptide:MHC II complex derived from it, we show that antigen-specific B cells residing in the follicles acquire antigen within minutes of injection, first in the region closest to the subcapsular sinus where lymph enters the lymph node. Antigen acquisition, presentation, and subsequent T cell-dependent activation did not require B cell migration through the T cell area or exposure to dendritic cells. These results indicate that the humoral response is initiated as soluble antigens diffuse directly from lymph in the subcapsular sinus to be acquired by antigen-specific B cells in the underlying follicles.