Homotypic cluster formation of dendritic cells, a close correlate of their state of maturation. Defects in the biobreeding diabetes-prone rat.

Aggregation of dendritic cells (DCs) in homotypic clusters has been described in vivo in lymph and skin, and here we report studies on homotypic clustering of rat splenic (s) DCs in vitro. Wistar rat sDCs readily formed homotypic clusters in culture, which increased in number and size over time (with a peak at t = 3 h). Keeping the cells at higher densities or treatment with anti-CD43 induced more and larger homotypic clusters. After such enhanced clustering the DCs had increased their T cell stimulating capabilities in syngeneic mixed lymphocyte reaction, and had a higher expression of CD80 and CD86 (signs of maturation). Ag transfer from bovine serum albumin-fluorescein isothiocyanate-pulsed to unpulsed DCs was observed during clustering. Here we also show that sDCs of the biobreeding diabetes-prone (BB-DP) rat, a model of autoimmune diabetes/thyroiditis, formed fewer and smaller clusters than Wistar sDCs, and that DC-DC clustering resulted in only a modest maturation of the cells (as determined in syn MLR and by phenotyping). Anti-CD43 completely restored the clustering defect BB-DP DCs in vitro, yet T cell-stimulating capability was only restored to a limited extent. Ag transfer in BB-DP DC clusters was similar.

A functional and phenotypic study on immune accessory cells isolated from the thyroids of Wistar and autoimmune-prone BB-DP rats.

Dendritic cells (DCs) comprise a small population of cells in the normal thyroid. These excellent antigen-presenting cells (APCs) are thought to be involved in the initiation of thyroid autoimmune reactions. However it is not known whether the APCs involved in this process are indeed DCs, or thyrocytes. Our aims were as follows: (1) to isolate DCs from the thyroid of normal Wistar rats and BB-DP rats prior to the development of lymphocytic thyroiditis; (2) to determine the T-cell stimulatory capability of such isolated thyroid DCs and to compare this capability to that of BB-DP thyrocytes and splenic DCs; and (3) to investigate the phenotype of isolated thyroid DCs and to compare it to that of splenic DCs; and (4) to investigate the capability of such thyroid DCs to regulate thyrocyte growth and function, and to compare it to our earlier reports demonstrating such capability with splenic DCs. Leukokcytic cell fractions were isolated from the thyroids of BB-DP and control Wistar rats of 7-20 weeks of age. The isolation steps included gentle enzymatic tissue disruption, the collection of non-plastic adherent cells and density gradient centrifugation of these cells to yield a low and a high density non-adherent fraction. The low density cell (LDC) fraction was composed of 50-75% leukocytes in both strains. These leukocytes were almost exclusively ED1+ monocytes or MHC-class II+ DC. The high density cell (HDC) fractions of both strains were composed of about 70% MHC-class II-negative thyrocytes and 30% ED1+ monocytes. The thyroid LDCs of both strains had an APC capability in syngeneic(syn)-MLR comparable to that of splenic DCs. However, the HDCs were extremely poor in syngeneic T cell stimulation. There was a difference in composition between the Wistar and the BB-DP LDC fractions: The Wistar LDCs were composed of 30-35% ED1+ monocytes and 15-20% typical MHC-II+ DCs, while BB-DP LDC fractions contained more ED1+ monocytes (about 70%), but fewer DCs (5-10%). In comparison to splenic DCs, thyroid DCs had a low CD80 and CD86 expression in both strains (i.e., an 'immature' phenotype). The LDCs of both animal strains were shown to decrease both basal and TSH-stimulated thyrocyte proliferation and T(3)release by about half. This report shows that a cell fraction enriched for monocytes and DCs can be isolated from the thyroids of both Wistar and BB-DP rats. The cells in this fraction were as capable as splenic DCs to act as T cell stimulators in syn-MLR. Since the thyroid HDCs (predominantly thyrocytes) were very poor at such T cell stimulation, thyroid monocytes and DCs (and not thyrocytes) are the prime candidates to act as immune accessory cells in the initiation of thyroid autoimmunity in the rat. Wistar thyroid LDCs differed in phenotype from BB-DP LDCs. The latter contained a lower percentage of DCs and a higher percentage of their precursors, the monocytes. Interestingly, a defect in the transition of monocytes to DCs has been described in another animal model of autoimmune thyroiditis/insulitis (the NOD mouse), as well as in thyroiditis and diabetic patients.

Signs of immaturity of splenic dendritic cells from the autoimmune prone biobreeding rat: consequences for the in vitro expansion of regulator and effector T cells.

From the biobreeding-diabetic prone (BB-DP) rat, an animal model for endocrine autoimmunity, phenotype and function of splenic dendritic cells (DC) were studied. Furthermore, the suppressive effect of peritoneal macrophages (pMphi) from the BB-DP rat in the MLR was investigated. Lower numbers of splenic DC were isolated from BB-DP rats than from control Wistar rats. In the preautoimmune phase, DC of the BB-DP rat had a lower surface MHC class II expression (and in preliminary data, a lower CD80 expression), ingested more bacteria, and had a lower stimulatory potency in the syngeneic (syn)MLR as compared with control DC. During disease development, the MHC class II expression further decreased, and a low stimulatory activity became evident in the allogeneic (allo)MLR. With regard to the expansion of suppressor/regulatory T cells, a lower percentage of RT6+ T cells but higher percentages of CD45RClow T cells were induced by BB-DP DC in synMLR, but not in alloMLR. An increase in the CD4/CD8 T cell ratio was observed in both the syn- and alloMLR due to a relative weak expansion of CD8+ T cells with DC of the BB-DP rat. Resident pMphi isolated from BB-DP or Wistar rats were equally effective in suppressing the DC-driven synMLR. In conclusion, splenic DC from the BB-DP rat have a lower accessory cell function already at young age, before the development of disease, and expanded different subsets of effector/suppressor T cells in vitro as compared with those from Wistar rats. The dysfunction of DC from BB-DP rats is likely to be caused by their relative immaturity as indicated by their low class II and costimulatory molecule expression and relatively high phagocytic activity.

Pre-autoimmune thyroid abnormalities in the biobreeding diabetes-prone (BB-DP) rat: a possible relation with the intrathyroid accumulation of dendritic cells and the initiation of the thyroid autoimmune response.

Thyroid autoimmune reactions start with an accumulation of mainly dendritic cells in the thyroid. There is increasing evidence that, apart from being antigen-presenting cells, they are also able to control the growth and hormone synthesis of neighbouring endocrine cells. The questions thus arise: are dendritic cells accumulating in the pre-autoimmune thyroid in response to an altered proliferative or metabolic activity of thyrocytes, and do cytokines, monocyte chemoattractants, or both, have a role in their accumulation? We have investigated these questions in thyrocytes of the biobreeding diabetes-prone (BB-DP) rat in relation to the start of the intrathyroid accumulation of dendritic cells--that is, at about 9 weeks of age. BB-DP rats and Wistar rats (controls) were studied from 3 to 20 weeks of age. Hyperplastic goitre development was studied by assessing the thyroid weight and by measuring the number of thyrocyte nuclei per 0.01 mm2 thyroid section. In addition, the in situ expression of interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-alpha), monocyte-chemotactic protein-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1) were studied by immunohistochemistry. The in vitro proliferative capacity of BB-DP and Wistar thyrocytes was measured by tritiated-thymidine ([3H]TdR) and bromodeoxyuridine (BrdU) incorporation into reconstituted, TSH- and non-TSH-stimulated, cultured thyroid follicles. Further in vitro studies consisted of measurement of the production of thyroxine (T4), triiodothyronine (T3), thyroglobulin, IL-6, TNF-alpha and MCP-1 by the thyroid follicles. BB-DP rats developed a small hyperplastic goitre between the ages of 9 and 12 weeks. The in vitro proliferative rate of thyrocytes isolated from hyperplastic BB-DP thyroids was significantly lower than that of Wistar thyrocytes. This phenomenon also occurred in follicles isolated from BB-DP rats before hyperplastic goitre development, which produced significantly less T4, but more T3, than did Wistar follicles of the same age. At the time of and after hyperplastic goitre development, BB-DP follicles exhibited altered metabolic behaviour and produced significantly more T4, but equal amounts of T3 compared with both Wistar follicles of the same age and follicles of younger BB-DP rats (both under basal conditions and TSH-stimulated). In vitro IL-6 production by these BB-DP thyroid follicles was also increased. There was no noteworthy difference in production of thyroglobulin and MCP-1 between BB-DP and Wistar follicles at any age. TNF-alpha was not produced by BB-DP or Wistar thyroid follicles. Immunohistochemistry revealed the expression of IL-6 by both BB-DP and Wistar thyroid follicle cells at all times of sampling. MCP-1 and TNF-alpha were expressed only when infiltrates were present in BB-DP thyroids (restricted to leucocytes, ages > 18 weeks). Modest ICAM-1 expression was restricted to large blood vessels in both BB-DP and Wistar thyroids; in the case of infiltrates (BB-DP rat) alone, high ICAM-1 expression was found on blood vessels and leucocytes in these infiltrations. At the time of intrathyroidal dendritic cells accumulation, BB-DP rats develop a small hyperplastic goitre. At that time there is also in vitro evidence for a shift to a higher production of thyroxine and IL-6 from thyrocyte follicles. The in vitro proliferation rate of BB-DP thyrocytes is, however, abnormally low (both in the pre- and hyperplastic period). Similar pre-autoimmune thyroid growth abnormalities have been described in another animal model of thyroid autoimmune disease, the obese strain chicken.

Antigen-presenting dendritic cells as regulators of the growth of thyrocytes: a role of interleukin-1beta and interleukin-6.

An accumulation of antigen-presenting dendritic cells (DC) in the thyroid gland, followed by thyroid autoimmune reactivity, occurs in normal Wistar rats during iodine deficiency, and spontaneously in diabetic-prone Biobreeding rats. This intrathyroidal DC accumulation coincides with an enhanced growth rate and metabolism of the thyrocytes, suggesting that both phenomena are related. Because DC are known to regulate the hormone synthesis and growth in other endocrine systems (i.e. the pituitary, the ovary, and the testis), we tested the hypothesis that DC, known for their superb accessory cell function in T cell stimulation, act as regulators of thyrocyte proliferation (and hormone secretion). We investigated the effect of (Nycodenz density gradient) purified splenic DC from Wistar rats on the growth rate of and thyroid hormone secretion by Wistar thyroid follicles (collagenase dispersion) in culture. Various numbers of DC and follicles were cocultured during 24 h. The proliferative capacity of thyrocytes was measured by adding tritiated thymidine (3H-TdR) and bromodeoxyuridine, the hormone secretion into the culture fluid was measured by using a conventional T3 RIA. Furthermore, antibodies directed against interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha) were added to these cocultures to determine the role of these cytokines in a possible DC regulation of thyrocyte growth. Cocultures were also carried out in the presence of antimajor histocompatibility complex-class I (MHC I), anti-MHC II, antiintercellular adhesion molecule-1 (ICAM-1), and antilymphocyte function-associated antigen-1alpha (LFA-1alpha) antibodies to possibly interfere with DC-thyrocyte interactions. The addition of DC to thyroid follicles clearly inhibited their 3H-TdR uptake, particularly at a 10:1 ratio, in comparison to follicle cultures alone, both under basal conditions and after TSH stimulation (75 +/- 7% and 49 +/- 11% reduction, respectively, n = 4). The follicle T3 secretion (after TSH stimulation) was also suppressed by DC in this system, but to a lesser extent (at best at an 1:1 ratio, 25 +/- 7% reduction, n = 4). The DC-induced inhibition of thyroid follicle growth was totally abrogated after addition of anti-IL-1beta antibodies; anti-IL-6 only had effect on the DC inhibition of non-TSH-stimulated thyrocytes, whereas anti-TNF-alpha demonstrated no effect at all. The antibodies to MHC and to adhesion molecules had also no effect on this DC-induced growth inhibition. The effect of the different anti-cytokine and anti-adhesion antibodies on the T3 secretion from thyroid follicles was not investigated. The clear inhibition of thyrocyte growth by splenic DC (classical antigen-presenting cells) again demonstrates the regulatory role of DC in endocrine systems. Proinflammatory cytokines such as IL-1beta and IL-6 are important mediators in this regulation. The here shown dual role of DC represents a link between the immune and endocrine system, which may form the gateway to the understanding of the initiation of thyroid autoimmune reactions and the thyroid autoimmune phenomena seen in iodine deficiency.

Reduced toxoplasmastatic activity of monocytes from AIDS patients: a role for granulocyte-macrophage colony-stimulating factor.

In the present study the role of granulocyte-macrophage colony stimulating factor (GM-CSF) in the reduced toxoplasmastatic activity of monocytes was investigated in patients with acquired immunodeficiency syndrome (AIDS). The secretion of GM-CSF by non-stimulated monocytes and by Toxoplasma gondii-infected monocytes from AIDS patients did not differ from that of healthy individuals. Furthermore, GM-CSF was not detected in sera from AIDS patients and healthy individuals. However, upon stimulation with lipopolysaccharide (LPS), monocytes from AIDS patients released significantly more GM-CSF than those from healthy individuals. Incubation of monocytes from AIDS patients with polyclonal antibodies against GM-CSF restored their inhibitory activity against T. gondii. On the basis of the present and earlier results the putative mechanism of reduced toxoplasmastatic activity of monocytes from AIDS patients may be as follows: upon stimulation with human immunodeficiency virus (HIV) the increased synthesis of GM-CSF by monocytes stimulates the production of prostaglandin E2 (PGE2) which, in turn, impairs the toxoplasmastatic activity of these cells.

Histomorphological aspects of the development of thyroid autoimmune diseases: consequences for our understanding of endocrine ophthalmopathy.

In this short review we will first evaluate the histomorphological aspects of the human and spontaneous animal thyroid autoimmune diseases. These diseases include Hashimoto goiter, primary myxedema, Graves' disease, and the spontaneous forms of thyroiditis in the Bio Breeding (BB) rat, the nonobese diabetic (NOD) mouse, and the obese strain (OS) of chicken. Based on sequential histomorphological events in the animal models of thyroid autoimmune disease, a mechanism for the pathogenesis of thyroid autoimmune disease is proposed. Since one of the human thyroid autoimmune diseases, specifically Graves' disease, is often associated with ophthalmopathy, the histomorphological aspects of the ophthalmopathic process are also evaluated to consider its possible autoimmune character.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) reduces toxoplasmastatic activity of human monocytes via induction of prostaglandin E2 (PGE2).

In this study we investigated the effect of human GM-CSF on the toxoplasmastatic activity and release of H2O2 and PGE2 by human monocytes. Incubation of monocytes from healthy controls with GM-CSF resulted in a dose-dependent reduction of toxoplasmastatic activity and a decrease in H2O2 production. Furthermore GM-CSF-treated monocytes released more PGE2 than untreated cells. To investigate the role of PGE2 in the reduced toxoplasmastatic activity of GM-CSF-treated monocytes, these cells were incubated with indomethacin. This resulted in a reduction of PGE2 release and restoration of toxoplasmastatic activity of monocytes treated with GM-CSF. GM-CSF reduces the toxoplasmastatic activity of monocytes via production of PGE2.

Reduced toxoplasmastatic activity of monocytes and monocyte-derived macrophages from AIDS patients is mediated via prostaglandin E2.

OBJECTIVE: To establish the role of prostaglandin E2 (PGE2) formed and released by monocytes and monocyte-derived macrophages (MDM) in the reduced toxoplasmastatic activity of these cells. DESIGN: Determination of PGE2 levels in the serum of AIDS patients, the release of PGE2 by monocytes and MDM from AIDS patients, the toxoplasmastatic activity of these cells and the effect of indomethacin, an inhibitor of PGE2 synthesis, on this cell function. SETTING: Laboratory of Cellular Immunology of the Department of Infectious Diseases, University Hospital, Leiden. PARTICIPANTS: Twenty-six AIDS patients. Healthy blood donors served as controls. RESULTS: The concentration of PGE2 in the serum from AIDS patients was significantly higher compared with serum from controls. Non-stimulated monocytes and lipopolysaccharide-stimulated monocytes and MDM from AIDS patients released significantly more PGE2 than corresponding cells from the controls. The proliferation of Toxoplasma gondii in monocytes and MDM from AIDS patients was significantly higher than in the respective cells from controls. Preincubation of these cells with indomethacin resulted in a decreased proliferation of T. gondii in non-activated monocytes and MDM and in interferon-gamma-activated MDM from AIDS patients. Preincubation of monocytes from healthy donors with PGE2 resulted in a dose-dependent increase of Toxoplasma proliferation which confirms that PGE2 can reduce the toxoplasmastatic activity of monocytes. CONCLUSION: PGE2 is involved in the reduced toxoplasmastatic activity of monocytes and MDM from AIDS patients.

Effect of IFN-gamma on the proliferation of Toxoplasma gondii in monocytes and monocyte-derived macrophages from AIDS patients.

This study was undertaken to determine whether the activity of monocytes and monocyte-derived macrophages (MDM) from acquired immune deficiency syndrome (AIDS) patients against Toxoplasma gondii is altered and whether this activity can be modulated by recombinant interferon-gamma (rIFN-gamma). Untreated and rIFN-gamma-treated monocytes or MDM from AIDS patients and from healthy controls were infected with T. gondii and the proliferation of these protozoa was determined. The H2O2 release by monocytes from AIDS patients and healthy controls was measured upon stimulation with phorbol myristate acetate (PMA) and formyl methionyl leucyl phenylalanine (FMLP). Monocytes from AIDS patients exhibited significantly lower toxoplasmastic activity compared to monocytes from healthy controls. The H2O2 release by monocytes from AIDS patients was also diminished. Incubation of monocytes from AIDS patients with rIFN-gamma for 2 days, but not 1 day, restored their toxoplasmastatic activity. The rate of proliferation of T. gondii was higher in MDM from AIDS patients than in MDM from healthy controls. Treatment of MDM from AIDS patients with rIFN-gamma for 1, 2 or 3 days resulted in partial inhibition of the proliferation of T. gondii. Collectively, these results demonstrate that the reduced toxoplasmastatic activity of monocytes and MDM from AIDS patients can be enhanced by in vitro treatment with rIFN-gamma, which supports the clinical use of rIFN-gamma for the treatment of opportunistic infections in these patients.

Development of antibody-forming cells in follicles of lymph nodes of mice after continuous antigenic stimulation.

It was the purpose of the present study to test a hypothesis on the direct differentiation of newly formed memory B cells into antibody-forming cells (AFC) in the follicles of lymph nodes. AFC may develop in follicles when mobile antigen is present at the moment that such memory B cells have completed their differentiation under influence of immune complexes trapped on follicular dendritic cells (FDC). In order to study whether the simultaneous presence of mobile antigen and immobilized immune complexes on FDC alters the normal distribution of AFC in lymph nodes, different immunization protocols with the thymus-dependent antigens trinitrophenylated keyhole limpet haemocyanin (TNP-KLH) and horseradish peroxidase (HRP) were used. Results confirm that, provided that mobile antigen and immobilized immune complexes are present simultaneously, AFC that are normally found in the medulla of the lymph node may also develop in the follicles.

Is early repopulation of macrophage-depleted lymph node independent of blood monocyte immigration?

Popliteal lymph nodes (LN) of mice were depleted of their macrophage (M phi) populations in the subcapsular sinus and medulla by subcutaneous injection of dichloromethylene diphosphonate (Cl2MDP)-containing liposomes into the footpads. Complete restoration of both M phi populations could be observed as late as 5 months after liposome administration. This relatively long repopulation time could be due to a depot of liposomes, directly killing all M phi precursors after extravasation into the interstitial tissue of the footpad. On the other hand, local interstitial precursors with very low turnover rates may have been depleted in the interstitial tissue of the hind leg. Therefore, two different types of experiments were performed; one in which M phi-depleted LN were replaced by control LN at various time points after liposome treatment, and another whereby M phi-depleted LN were transplanted into control animals. When liposome-treated, M phi-depleted LN were transplanted into control animals, a complete restoration of both populations in the subcapsular sinus and medulla could be observed within 5 weeks. Control LN transplanted into a Cl2MDP-liposome-treated leg showed a rapid disappearance of M phi from the subcapsular sinus and medulla and these cell populations remained absent for at least 7-8 weeks after liposome treatment, when the first cells started to reappear. Complete repopulation of these areas by M phi took as long as 15 weeks. Using labeled liposomes the presence of a continuous liposome depot was found to be very unlikely. These results suggest that the population of precursor cells that will give rise to M phi in the subcapsular sinus and medulla of a LN is probably contained within the interstitial tissue and is almost independent of precursor supply from the blood compartment.

Elimination of spleen and of lymph node macrophages and its difference in the effect on the immune response to particulate antigens.

To study the role of macrophages in the in situ immune response to particulate antigens in spleen and popliteal lymph nodes (PLN), mice were injected with dichloromethylene diphosphonate (Cl2MDP)-containing liposomes to eliminate macrophages, followed by immunization with trinitrophenylated sheep red blood cells (TNP-SRBC). Depletion of macrophages in the spleen caused a strong decrease in the number of antibody-forming cells (AFC), which develop after intravenous (i.v.) injection of the antigen. These results strongly suggested the involvement of splenic macrophages in the processing of TNP-SRBC. In particular, the populations of marginal zone macrophages may be involved in the inductive phase of an antibody response to particulate antigens. These macrophages are strategically positioned at the end of the white pulp capillaries in the marginal zone of the spleen and they have their cell processes between the marginal zone-B cells. Elimination of macrophages in PLN had no effect on the number of AFC, which develop after subcutaneous (s.c.) injection of the antigen in the hind footpads. This indicates that the macrophages are not essential for the induction of a local immune response to the particulate antigen TNP-SRBC. After depletion of lymph node macrophages, the number of AFC developing in the spleen after s.c. footpad injection of the antigen increased and the anti-TNP serum titers were elevated. This may well be caused by the fact that more of the antigen reaches the circulation and subsequently stimulates the spleen.

The in situ immune response in popliteal lymph nodes of mice after macrophage depletion. Differential effects of macrophages on thymus-dependent and thymus-independent immune responses.

Mice were subcutaneously (SC) injected in the left hind footpad with dichloromethylene diphosphonate (Cl2MDP)-containing liposomes to eliminate macrophages lining the subcapsular sinus (SCS) and those in the medulla of draining popliteal lymph nodes (PLN). In order to study the effect of depletion of these macrophages on the in situ immune response in the PLN, liposome-treated mice were SC injected in the same footpad with thymus-independent (TI) type 1 antigen trinitrophenylated lipopolysaccharide (TNP-LPS), TI-type 2 antigen TNP-Ficoll or thymus-dependent (TD) antigen TNP-keyhole limpet haemocyanin (TNP-KLH). No major differences were observed in antibody-serum titers of liposome-treated and control animals. After primary as well as secondary immunization with the TD-antigen TNP-KLH, an increase in the number of antibody-forming cells (AFC) was found and the peak of response was delayed in the PLN of liposome-treated animals. Such differences were not observed with the TI-antigens. These results indicate that macrophages lining the SCS and those in the medulla of the PLN are not essential for the induction of an immune response. The positive effect of macrophage-depletion on the number of AFC may be explained by competition for the antigen by macrophages and other antigen-presenting cells.

Repopulation of macrophages in popliteal lymph nodes of mice after liposome-mediated depletion.

Macrophages lining the subcapsular sinus (SCS) and those located in the medulla of popliteal lymph nodes (PLN) of mice were eliminated after subcutaneous (s.c.) injection of dichloromethylene diphosphonate (Cl2MDP)-containing liposomes. No effect of liposome-entrapped Cl2MDP could be seen on nonphagocytic cells, e.g., interdigitating cells (IDC) and B- and T-lymphocytes. One month after injection the eliminated subsets of macrophages were still absent. After 2 mo a small number of macrophages had reappeared along the SCS and in the medulla of the PLN of a few animals. Complete repopulation of the PLN with macrophages was observed only after 5 mo. This extremely long repopulation time could be shortened drastically by local administration of Freund's complete adjuvant (FCA). A small number of macrophages reappeared along the SCS and in the medulla 1 wk after FCA and after 3 wk the repopulation of the PLN with macrophages was complete. Such rapid repopulation of macrophages was not achieved after s.c. injection of paraffin oil or paratyphoid vaccine. These results indicate that the normal rate of influx of mononuclear phagocytes into the PLN is low, but that it can be sped up after administration of FCA.

Primary in situ immune response in popliteal lymph nodes and spleen of mice after subcutaneous immunization with thymus-dependent or thymus-independent (type 1 and 2) antigens.

Mice were immunized subcutaneously with thymus-independent (TI)-type 1 antigen trinitrophenylated lipopolysaccharide (TNP-LPS), TI-type 2 antigen TNP-Ficoll or thymus-dependent (TD) antigen TNP-keyhole limpet haemocyanin (TNP-KLH) in order to study the primary in situ immune response in popliteal lymph nodes (PLN) and spleen. The spleen responded more rapidly in developing specific antibody-forming cells (AFC) than the lymph nodes did, in spite of the fact that antigens reach the spleen only after passing several lymph node stations. This difference between lymph nodes and spleen in developing AFC was particularly significant with respect to the responses to TI (both type 1 and type 2) antigens. No differences in the distribution of specific AFC in PLN and spleen were observed after immunization with TI and TD antigens. Results are discussed with respect to the relative contributions of lymph nodes and spleen to immune responses to antigens injected subcutaneously.