Elimination of phagocytic cells in the spleen after intravenous injection of liposome-encapsulated dichloromethylene diphosphonate

Summary Dichloromethylene diphosphonate can be used for temporary elimination of macrophages in the spleen when administered after entrapment in liposomes. No comparable effect on the macrophages of the spleen was observed with free dichloromethylene diphosphonate or in the case of empty liposomes. Marginal metallophils on the boundary between white pulp and marginal zone as well as macrophages in the marginal zone and red pulp disappeared from the spleen within one day and remained largely absent for about a week. After this time cells reappeared slowly, and at approximately four weeks after injection their presence in the spleen did not differ from that in control animals. Marginal metallophils and macrophages in the spleen were demonstrated by use of enzyme-histochemical methods and by their capacity to ingest carbon particles.     

Influence of lymphocytes on the presence and organization of dendritic cell subsets in the spleen.

Studies were undertaken to clarify the roles of individual leukocyte populations in maintaining the presence and organization of splenic dendritic cells (DCs). Using Abs specific for DC subsets, we found that the distinct types of DC maintained appropriate compartmentalization within the white pulp of lymphocyte-deficient mice despite an unusual overall distribution of DCs. Even in mice lacking both B and T lymphocytes, the central arteriole remained the structure around which T area DCs were organized. Marginal zone area DCs remained in a peripheral sheath excluded from the T area DCs. Additionally, we revealed an important role for splenic B cells in the presence and organization of marginal zone cells. B-deficient or B- and T-deficient mice lacked sialoadhesin+ marginal zone macrophages and lacked MAdCAM-1 expression in marginal zone reticular endothelial cells. Adoptive transfer of B lymphocytes induced MAdCAM-1 expression but failed to recruit marginal zone macrophages. Taken together, our results demonstrate that the arrival, localization, and persistence of DCs in spleen are events not solely dependent upon signals from the mature B and T cells or marginal zone macrophages. We suggest that specific stromal elements in the vicinity of the central arteriole are primarily responsible for providing directional cues to the DC.     

Plasma membrane lipid organization and the adherence of differentiating lymphocytes to macrophages

Changes in the packing of phospholipids in the plasma membrane of lymphocytes occur during differentiation within primary and secondary lymphoid organs. As they differentiate, lymphocytes interact with a variety of reticuloendothelial cells, including macrophages. To investigate a possible relation between these two phenomena, the strength of the interactions between lymphocytes and macrophages was measured in vitro as a function of the tightness of packing of phospholipids on the lymphocyte surface. Strength of adherence was measured by the ability of lymphocytes to remain adherent to macrophages when subjected to increasing centrifugal forces. Phospholipid packing was assessed using the fluorescent lipophilic probe merocyanine 540 (MC540), which preferentially binds to bilayers in which the lipids are more loosely packed. Three subpopulations of murine thymocytes were resolved with respect to strength of adherence to peritoneal or thymic macrophages. To determine whether these subpopulations corresponded with the three classes of cells distinguishable by MC540 fluorescence, populations enriched for staining or non-staining cells, and cells sorted on the basis of MC540 fluorescence intensity, were examined. The least fluorescent cells were the least strongly adherent; the most fluorescent cells were the most strongly adherent; and cells of intermediate fluorescence had intermediate adherence. When splenic lymphocytes were examined with respect to adherence to peritoneal or splenic macrophages, similar patterns of fluorescence and adherence were seen. These results suggest that the organization of the plasma membrane lipid bilayer of lymphocytes may be involved in their interactions with macrophages during primary and secondary differentiation. The adherence signal for lymphocytes thus may be similar to that proposed for other blood cells.     

Germinal centers in the human thymus

Summary This study reports the normal thymus of a 3 year old girl in which four germinal centers have been found in different lobules. They are located in the medulla, three of them in the cortico-medullary junction. Reticular cells, medium-sized and occasionally large lymphocytes, reticular macrophages and a large number of immature and mature thymocytes occupy the germinal centers. The medium-sized lymphocytes divide and give rise to smaller cells which appear to move to the peripheral zone to become thymocytes.     

The requirement for macrophage-lymphocyte interaction in T lymphocyte proliferation induced by generation of aldehydes on cell membranes.

Guinea pig T lymphocyte proliferation induced by sodium periodate (NaIO4) or neuraminidase-galactose oxidase (NG) occurs when lymphocytes and macrophages are cultured together after treatment of either purified T lymphocytes or macrophages with these agents. Regardless of which cell initially bears the modified surface carbohydrate, lymphocyte proliferation requires the presence of viable homologous macrophages and fails to occur when they are replaced with fibroblasts, erythrocytes, L2C leukemia cells, thymocytes, PMN, line I hepatoma cells, or murine macrophages. Lymphocyte proliferation resulting from NaIO4 or NG treatment of lymphocytes is diminished when these cells are treated with proteolytic enzymes or aged in in vitro culture for 48 hr. By contrast, proteolytic enzyme treatment or in vitro aging has no effect on the ability of NaIO4 or NG-treated macrophages to induce lymphocyte proliferation. The requirement for macrophage-lymphocyte interaction in NaIO4 or NG-induced lymphocyte proliferation is indicative of a central role for the macrophage in the initiation of T lymphocyte proliferation.     

Morphology and distribution of cystic cavities in the normal murine thymus

Summary The normal murine thymus was examined by lightand electron microscopy to determine the distribution and morphology of extracellular cystic cavities. Most cavities were confined to the cranial half of each gland, situated at the junction between cortex and medulla. They varied in size and shape, and gave rise to narrow channels that coursed to the capsular surface of the gland. Large cavities could be divided into three zones. A short cranial zone exhibited gland-like features, consisting of cells lining a clear lumen. A central zone was lined by a diverse population of cells. Some possessed secretory granules, while others exhibited an apical ciliated border. Lining cells interdigitated with each other and were joined laterally by intercellular junctions. The lumen of the central zone contained lymphocytes and macrophages in an amorphous extracellular matrix. The caudal zone of each cavity had an attenuated and incomplete cellular lining, communicating directly with the surrounding thymic parenchyma. Thymic cavities may represent the initial part of the efferent lymphatic system of the gland, beginning in the tissue spaces at the corticomedullary junction. Selected cells could then enter and interact with the luminal contents in the central zone of the cavity. Ciliated cells may then propel lymphocytes and secretions into the narrow channels radiating from the uppermost part of the chamber, leaving a cell-free lumen in this region. These cavities may function in sequestering lymphocytes, macrophages and thymic secretions before their exit from the gland.     

Mise en évidence des cellules inflammatoires dans le sperme

A high level of round cells in semen decreases the fertility capacity. These cells can be either immature germinal cells from testicular disorders characterized by teratospermia, or polymorphonuclear cells if they are peroxidase positive. However it often happens that neither teratospermia nor peroxidase reaction can explain the high proportion of round cells. For these semen we have used the nonspecific esterase staining to search for macrophages, plasma-cells and lymphocytes. These three types of cells were detected in certain non-leucospermic semen which reflects a chronic infection in development. Also these cells, macrophages, plasma-cells and lymphocytes were found in some leucospermic semen samples. If we considere the populations leucospermic and non leucospermic together, macrophages are correlated — with plasma-cells (r=0,65 — p=0,01) and with lymphocytes (r=0,56 — p=0,05). The correlation between plasma-cells and lymphocytes was more important (r=0,91 — p=0,001).     

Ultrastructure of splenic white pulp of the turtle,Mauremys caspica

Summary The ultrastructure of splenic tissue of non-immunized turtles, Mauremys caspica, shows two areas, namely, the white pulp which is lymphoid in nature, and the red pulp which is formed by cell cords and sinusoids. Between both areas there is always a marginal zone with gaps through which cells leak. In the white pulp, there are two blood vessel types; one with muscled walls, and the other showing thinner walls sheathed by reticular cells. Reticular cells constitute a network where there occur dendritic macrophages, lymphoblasts and small and medium lymphocytes. Mature plasma cells are scarce in the white pulp.     

Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages.

During normal tissue remodeling, macrophages remove unwanted cells, including those that have undergone programmed cell death, or apoptosis. This widespread process extends to the deletion of thymocytes (negative selection), in which cells expressing inappropriate Ag receptors undergo apoptosis, and are phagocytosed by thymic macrophages. Although phagocytosis of effete leukocytes by macrophages has been known since the time of Metchnikoff, only recently has it been recognized that apoptosis leads to surface changes that allow recognition and removal of these cells before they are lysed. Our data suggest that macrophages specifically recognize phosphatidylserine that is exposed on the surface of lymphocytes during the development of apoptosis. Macrophage phagocytosis of apoptotic lymphocytes was inhibited, in a dose-dependent manner, by liposomes containing phosphatidyl-L-serine, but not by liposomes containing other anionic phospholipids, including phosphatidyl-D-serine. Phagocytosis of apoptotic lymphocytes was also inhibited by the L isoforms of compounds structurally related to phosphatidylserine, including glycerophosphorylserine and phosphoserine. The membranes of apoptotic lymphocytes bound increased amounts of merocyanine 540 dye relative to those of normal cells, indicating that their membrane lipids were more loosely packed, consistent with a loss of membrane phospholipid asymmetry. Apoptotic lymphocytes were shown to express phosphatidylserine (PS) externally, because PS on their surfaces was accessible to derivatization by fluorescamine, and because apoptotic cells expressed procoagulant activity. These observations suggest that apoptotic lymphocytes lose membrane phospholipid asymmetry and expose phosphatidylserine on the outer leaflet of the plasma membrane. Macrophages then phagocytose apoptotic lymphocytes after specific recognition of the exposed PS.     

Studies on the regulation of lymphocyte reactivity by normal and activated macrophages.

To determine the potential role of macrophages as regulators of the immune response, the effect of mouse peritoneal macrophages on transforming mouse spleen lymphocytes was investigated. Mitogen and antigen stimulated lymphocyte transformation, as measured by DNA synthesis, was enhanced by all concentrations of normal macrophages tested, but only by low concentrations of activated macrophages. High concentrations of activated macrophages markedly inhibited lymphocyte transformation. This inhibition occurred whether lymphocyte DNA synthesis was measured by incorporation of [³H]TdR or of ³²P. Activated macrophages cultured with lymphocytes within 4 hr of being removed from the peritoneal cavity inhibited lymphocyte transformation. When activated macrophages were cultured alone for 24 or more hours before addition of lymphocytes, enhancement of transformation was noted. Once lymphocytes were exposed to activated macrophages, they could not be induced to undergo transformation in the presence of Con A. Whereas heat-killed activated macrophages, which appeared intact morphologically, lost their capacity to inhibit lymphocyte transformation, macrophages treated with mitomycin C to inhibit DNA synthesis retained this capacity. Syngeneic and allogeneic macrophages had similar inhibitory ability. Supernatants from cultures of many cell types (including normal or activated macrophages, lymphocytes, lymphocytes plus macrophages, and L cells) inhibited [³H]TdR incorporation by both mitogen stimulated lymphocytes and tumor cells. These studies demonstrate the capacity of macrophages to regulate lymphocyte transformation in vitro and suggest a role for these cells as regulators of cell-mediated immunity in vivo.     

Macrophage-lymphocyte clusters in lymph nodes: a possible substrate for cellular interactions in the immune response.

The lumens of the lymphatic sinuses in lymph nodes are traversed by fibrocellular trabeculae. Joined to these trabeculae were macrophages, which formed cell clusters with lymphocytes. It is proposed, based on structural similarities, that these cell clusters are the equivalent in vivo to those seen during primary and secondary immune responses in vitro. These intraluminal macrophages were located in the path of lymph-borne antigen, as well as in the path of newly formed and recirculating lymphocytes in sinuses. This would facilitate the possible interaction between macrophage-associated antigen and antigen-reactive lymphoid cells. The attachment of numerous lymphocytes to the surfaces of macrophages and the resulting cell clusters also afford increased opportunities for lymphocyte-lymphocyte contact.     

AN ELECTRON MICROSCOPE STUDY OF LYMPHATIC TISSUE IN RUNT DISEASE

The thymus, spleen, and lymph nodes were studied in runt disease induced by a graft of intravenously injected homologous splenic cells into newborn rats and mice. Adult Long-Evans cells (70 x 10⁶) were injected into Sprague-Dawley rats. Adult DBA cells (7 x 10⁶) were injected into C57BL/6 mice. Runted rats were sacrificed at 14 to 28 days of age; mice at 10 to 20 days. The thymic cortex is depleted of small lymphocytes. Those remaining are severely damaged and phagocytized. Evidence of damage includes swelling of mitochondria, myelin figure formation, margination of chromatin, and sharp angulation in nuclear contour. Large numbers of macrophages are present. Epithelial-reticular cells which envelop small cortical blood vessels are often retracted, with the result that the most peripheral layer in the thymic-blood barrier suffers abnormally large gaps. Lymphocytes of the periarterial lymphatic sheaths of spleen and of the cortex of lymph nodes are reduced in number and damaged. Vast numbers of plasma cells and many lymphocytes are evident throughout lymph nodes, in the periarterial lymphatic sheaths, and in the marginal zone and red pulp of the spleen. Plasma cells are of different sizes, the larger having dilated sacs of endoplasmic reticulum. Lymphocytes are small to medium in size. They contain, in varying quantity, ribosomes and smooth membrane-bounded cytoplasmic vesicles approximately 350 to 500 A in diameter. Most plasma cells and lymphocytes are damaged and many of these are phagocytized. Many lymphocytes in lymph nodes, however, show no evidence of damage. Reticular cells and other fixed cells of the connective tissues seldom appear affected. Thus, the major cell types reacting in runt disease are lymphocytes, plasma cells, and histiocytes or macrophages. It appears, therefore, that both the delayed and immediate types of sensitivity play a part in this disease.     

Macrophage infiltration in tumors and tumor-surrounding tissue: influence of serotonin and sensitized lymphocytes

Summary In a delayed-type hypersensitivity reaction serotonin released from mast cells plays an important role in the induction of a cellular infiltrate at the site of antigen challenge. In analogy, we have studied whether it is possible to enhance the number of intratumoral macrophages by injecting serotonin into a s.c. SL2 lymphosarcoma. The vessels in the tissue surrounding the tumor responded well to serotonin, as there was an influx of i.v. injected ⁵¹Cr-labeled sensitized spleen cells in this tissue during the first 4 h after intratumoral injection of serotonin. At 24 h after serotonin injection there was an influx of macrophages into this tumor-surrounding tissue. No influx of cells was detected in the tumor itself during the first hours after injection of serotonin. In the tumor, similar phenomena occurred as in the surrounding tissue, but with a delay of about 24 h. This suggests that lymphocytes leave the blood circulation in the tumor-surrounding tissue and migrate to the tumor. The influx of macrophages into the tumor after intratumoral injection of serotonin is probably due to an immunological reaction as the lymphocyte influx preceeds the macrophage influx into tumors. In addition, transfer of sensitized lymphocytes, as well as lymphocytes from a tumor-bearing host caused an enhanced influx of macrophages into the tumor. To test the specificity and serotonin dependency of the phenomenon of infiltrating cells in tumors we have used a footpad swelling assay in which the serotonin dependency and the antigen specificity of the response against syngeneic tumor cells was shown. The following picture emerged: an intratumoral serotonin injection enables lymphocytes to leave blood vessels in the tumor-surrounding tissue. These lymphocytes with specificity for tumor antigens migrate to the tumor. After contact with the antigenic tumor cells, these lymphocytes secrete chemoattractive factors for monocytes/macrophages. Also these monocytes/macrophages leave the circulation in the tumor-surrounding tissue. Subsequently the macrophages invade the tumor. We conclude that the number of intratumoral macrophages can be enhanced by serotonin.     

Pathogenesis of experimental allergic orchitis. III. T lymphocyte requirement in local adoptive transfer by peritoneal exudate cells.

In experimental allergic orchitis (EAO), a lesion characterized by mononuclear invasion of seminiferous tubules can be adoptively transferred within 1 to 4 days by testicular injection of peritoneal exudate cells (PEC) from syngeneic strain 13 guinea pigs (GP) immunized with homologous testicular antigens in complete Freund's adjuvant (CFA). This study examined the role of T lymphocytes, macrophages, and polymorphonuclear neutrophils (PMN) in the adoptive transfer. Guinea pig PEC contained 7% T lymphocytes, rare B lymphocytes, and over 90% of macrophages and PMN. After T lymphocytes were depleted by rabbit erythrocyte (E) rosette and Hypaque-Ficoll gradient centrifugation, cell preparations that contained 73% of original macrophages and 15% original T lymphocytes were obtained, and these cells did not transfer EAO (0 of 18 testes). In contrast, cell preparations enriched in T lymphocytes by nylon wool column or E rosette contained 1.5% of the original macrophages and 59% of the original T lymphocytes transferred EAO to 70% of the testes, starting at 1.5 x 10(6) T lymphocytes per testis. The number of T lymphocytes correlated with the incidence of adoptive transfer; the correlation existed regardless of the number of macrophages or PMN present. Finally, EAO was adoptively transferred to recipients that had total-body irradiation. The results indicate that (a) T lymphocytes are capable of transferring lesions of EAO, (b) in the transfer, the T lymphocytes did not function as helper T cells, since the transfer need not involve participation of host lymphoid cells, and (c) by inference, testis antigen-reactive T lymphocytes exist.     

Suppressor macrophages in lymphocyte proliferation of cynomolgus monkeys

The present study demonstrated the presence of cells belonging to monocyte/macrophage lineage which suppressed mitogen-induced blastogenesis of peripheral blood lymphocytes in cynomolgus monkeys. Depletion of adherent or phagocytic cells from peripheral mononuclear cells caused a substantial increase in the blastogenic response of cynomolgus monkey lymphocytes whereas the same treatment led to marked reduction rather than enhancement in human lymphocyte blastogenesis. Addition of thioglycollate-elicited peritoneal exudate adherent cells as macrophages suppressed the blastogenic response of nonadherent lymphocytes in a dose-dependent manner. The suppressive effect was observed not only in autologous but also in allogeneic macrophages to the responder lymphocytes. Treatment of macrophages with silica, carrageenan or freezing-thawing reduced their suppressive effect but there was no reduction with mitomycin C or indomethacin. No suppressive activity was detected in the cell-free supernatant of macrophages cultured in the presence or absence of mitogens for up to 4 days. From these findings, it appeared that monocyte/macrophage lineage might be responsible for the observed suppressive effect on mitogen-induced blastogenesis of cynomolgus monkey lymphocytes.     

The fine structure of the cells of the mouse peritoneum

Summary The cells of the peritoneum of the mouse have been examined with the electron microscope both by studying the gastro-splenic omentum and by washing the cells out of the peritoneal cavity. They comprise mesothelial cells, mast cells, lymphocytes and macrophages. The mesothelial cells were probably nearly all degenerate. The mast cells released granules which were phagocytosed by the other cells. The lymphocytes were either classical small lymphocytes, or rather larger cells similar to previously described immunoblasts. The macrophages varied considerably in size. Some were probably derived from the covering cells of the milk spots. They contained varying numbers of dense bodies, with the structure of lysosomes. A series of appearances was seen which suggested that these were synthesized in the granular endoplasmic reticulum. A gradation of structure was seen between lymphocytes and small macrophages.The gastro-splenic omentum consisted of two layers of mesothelium, in places fenestrated. The milk spots which were scattered throughout this structure were covered by cells similar to macrophages, and had a core of lymphoid cells in which ran a small blood vessel. The most notable difference between the mesothelial cells and the macrophages was the presence of many small caveolae at the surface of the mesothelial cells, and of larger vacuoles and indentations at the surface of the macrophages. Acknowledgements. I am grateful to Professor R. Barer for much advice and criticism, to Dr. G. A. Meek for guidance on electron microscopy, and to Miss M. Tune and Mr. M. Turton for photographic assistance.This work was supported by a grant from the Medical Research Council and by grants to the Department from the S.R.C., Nuffield Foundation and Unilever Limited.     

Changes in localization of immune cells and cytokines in corpora lutea during luteolysis in murine ovaries

Immune cells, which constitute a significant cell mass in the corpora lutea (CLs), are considered to play critical roles in luteolysis, but the details are not fully understood. We histochemically investigated the changes in distribution and cell density of macrophages and T lymphocytes and in tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma, which can induce apoptosis in the luteal cells in murine CLs during luteal regression. No macrophages or T lymphocytes were observed in functionally healthy CLs. Abundant macrophages and increasing T lymphocytes were demonstrated in CLs at the functional regression stage (early stage of regression). At the structural regression stage (late stage of regression), abundant T lymphocytes but no macrophages were demonstrated in the CLs. A moderate amount of TNF-alpha was detected in all CLs at all stages. No IFN-gamma was detected in either healthy or early regressing CLs, but a large amount of IFN-gamma was detected at the late regression stage. Moreover, in cultured luteal cells, reactivity against Fas-ligand (FasL) was caused by pretreatment with TNF-alpha and IFN-gamma and apoptosis was induced by FasL treatment. These findings support the hypothesis that macrophages initiate T lymphocyte aggregation at the early stage of luteal regression, and then T lymphocytes induce apoptosis on luteal cells, which in turn develop sensitivity against FasL by TNF-alpha and IFN-gamma.     

Ultrastructural and histochemical investigation of the terminal capillaries in the spleen of the carp (Cyprinus carpio L.)

Summary In the spleen of the carp arterial capillaries of a highly differentiated structure have been studied by light and electron microscopy. These capillaries share various structural characteristics with the sheathed capillaries (ellipsoids of Schweigger-Seidel) of higher vertebrates. The long arterial capillaries of the carp spleen are provided with cuboidal endothelial cells containing filaments approximately 7 nm in diameter. There is no basal lamina. The endothelial cells form various types of cell junctions, but there are also extensive areas without any junctions. Here, a free passage is possible between the capillary lumen and the subendothelial space. The capillaries possess a single-layered sheath of macrophages. Characteristically, the sheath macrophages possess long and slender cell processes forming a loose framework, the meshes of which are filled with lymphocytes and spindle cells. The sheath macrophages show a zone of ectoplasm rich in filaments. They also contain numerous phagolysosomes rich in hydrolytic enzymes, as identified histochemically. The sheath is sharply limited against the pulp by a thick layer of collagen fibers.     

Infiltration of H-2d-specific cytotoxic macrophage with unique morphology into rejection site of allografted meth A (H-2d) tumor cells in C57BL/6 (H-2b) mice

It is assumed that CD8(+) cytotoxic T lymphocytes (CTLs) mediate direct lysis of allografts and that their growth, differentiation, and activation are dependent upon cytokine production by CD4(+) helper T lymphocytes. In the present study, the effector cells responsible for the rejection of i.p. allografted, CTL-resistant Meth A tumor cells from C57BL/6 mice were characterized. The cytotoxic activity was associated exclusively with peritoneal exudate cells and not with the cells in lymphoid organs or blood. On day 8, when the cytotoxic activity reached a peak, 3 types of cells (i.e., lymphocytes, granulocytes, and macrophages) infiltrated into the rejection site; and allograft-induced macrophages (AIM) were cytotoxic against the allograft. Bacterially-elicited macrophages also exhibited cytotoxic activity (approximately 1/2 of that of AIM) against Meth A cells, whereas the cytotoxic activity of AIM against these cells but not that of bacterially-elicited macrophages was completely inhibited by the addition of donor (H-2(d))-type lymphoblasts, suggesting H-2(d)-specific cytotoxicity of AIM against Meth A cells. In contrast, resident macrophages were inactive toward Meth A cells. Morphologically, the three-dimensional appearance of AIM showed them to be unique large elongated cells having radiating peripheral filopodia and long cord-like extensions arising from their cytoplasmic surfaces. The ultrastructural examination of AIM revealed free ribosomes in their cytoplasm, which was often deformed by numerous large digestive vacuoles. These results indicate that AIM are the H-2(d)-specific effector cells for allografted Meth A cells and are a more fully activated macrophage with unique morphological features.     

Macrophage-T cell interaction mediated by immunogenic and non-immunogenic forms of a monofunctional antigen

Summary As an approach to the elucidation of the essential steps in the immune pathway, the uptake and retention of immunogenic and non-immunogenic analogs of a monofunctional antigen by guinea pig macrophages and the efficiency of macrophages pulsed with the compounds to present antigen to sensitized T lymphocytes were compared. L-Tyrosine-azobenzene-p-arsonate (RAT) and its non-immunogenic analog, 4-hydroxyphenyl-n-propane-3-azobenzene-p-arsonate (RAN), react similarly with antiarsonate antibody, but RAN, unlike RAT, is unable to induce cellular immunity in guinea pigs. The uptake and retention patterns of the two compounds by macrophages differed in that, at a given time, more RAN than RAT was retained and detectable on cell surfaces by anti-arsonate antibody. Equivalent numbers of T lymphocytes from guinea pigs sensitized to RAT formed antigen-dependent clusters with macrophages pulsed with either RAT or RAN after 24 hr in culture, but not with macrophages pulsed with an azobenzenoid compound of unrelated specificity. On the other hand, T lymphocytes from guinea pigs immunized with RAN showed no significant capacity to bind to macrophages which had been pulsed with any of the compounds. The number of lymphocytes from RAT-sensitized animals which bound to RAT-pulsed macrophages remained relatively stable over a 48 hr period, whereas clusters of the same lymphocytes with RAN-pulsed macrophages dissocitated to background levels within that time. Early cluster formation mediated by RAN, as well as its ability to induce transient specific T cell unresponsiveness to RAT in vivo, indicate that T cells are capable of recognizing (binding) the non-immunogen. However, such early, and perhaps weak, interaction with RAN-pulsed macrophages did not induce DNA synthesis by T cells. Anti-Ia serum completely blocked cluster formation mediated by either RAT or RAN. Thus, the only significant distinction disclosed by these studies between the immunogenic and non-immunogenic compounds was the stability of macrophage-T cell interaction as determined by the persistence of antigen mediated cell clusters in culture, suggesting that this may be a factor in immunogenic discrimination.Abbreviations ABA azobenzenearsonate - BSA bovine serum albumin - CFA complete Freund's adjuvant - IFA incomplete Freund's adjuvant - KLH keyhole limpet hemocyanin - LNC Lymph node cells - MHC major histocompatibility complex - PEC peritoneal exudate cells - PEL peritoneal exudate lymphocytes - RAN 4-hydroxyphenyl-n-propane-3-azobenzene-p-arsonate - RAT L-tyrosine-azobenzene-p-arsonate - TAT L-tyrosine-azobenzene-p-trimethylammonium chloride Aided by USPHS Grant AI 05664.