Antibody OKT3 Research Articles

Dissection of the role of macrophages in triggering T lymphocytes for interleukin 2 production by monoclonal antibody OKT3.

Unfractionated human peripheral blood mononuclear cells produce a small amount of interleukin 2 (IL 2) by stimulation with a monoclonal anti-T3 antibody (OKT3) in vitro. The IL 2 production could be greatly augmented by the addition of a phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). In the presence of TPA, the T cell enriched fraction deprived of macrophages did not produce IL 2, but the T cells pulse-incubated with OKT3 and reconstituted with macrophages efficiently produced IL 2 in subsequent culture in the presence of TPA as did T cells reconstituted with OKT3-pulse-incubated macrophages. The stimulating effect of OKT3 in the presence of macrophages was inhibited dose-dependently by the addition of immunoglobulins, particularly by mouse IgG2a which is the same isotype as that of the OKT3 antibody, showing that it inhibits by blocking the binding of OKT3 to Fc receptors on macrophages. The same extent of IL 2 production was induced in T cells when paraformaldehyde-fixed macrophages were substituted for intact macrophages. Remarkable IL 2 production was also induced by OKT3 when latex beads coated with rabbit anti-mouse IgG2a antibody and TPA were added to the culture. It was confirmed that the production induced by these stimulations was due to an increase of IL 2 mRNA. These results show that effective signals for IL 2 production are generated by efficient crosslinking of T3 molecules which results from multi-interaction of T3 molecules on the T cell membrane and anti-T3 antibody molecules on macrophage membrane or on the surface of the latex particle.

A standard microcytotoxicity technique for quantitative analysis of lymphocyte subsets: A comparison with indirect immunofluorescence, evaluated by microscopy or flow cytometry

A standard complement-dependent microcytotoxicity (CDC) technique was used for quantitative analysis of T-lymphocyte subsets in human peripheral blood and the results compared to those obtained by indirect immunofluorescence microscopy and flow cytometry. The monoclonal antibodies OKT3, OKT4 and OKT8 were used in the CDC method for detection of total-T cells, T-helper and T-suppressor cells respectively. The CDC technique provided reproducible results (CV, 3–7%) correlating well with both immunofluorescence techniques. This observation was valid both for healthy persons ( n = 21) and for patients ( n = 10) with immunological disorders. The correct antibody dilution, correction for background and the use of eosin staining are considered critical for the usefulness of this technique. The method has several advantages: it is widely used for histocompatibility testing, only simple equipment is necessary, and the amount of monoclonal antibody required per test is small.

Results in two infants with the DiGeorge syndrome—Effects of long-term TP 5

We treated two patients affected by DiGeorge syndrome with long-term administration of the synthetic thymic hormone thymopoietin (TP 5). In both cases we obtained durable immunological reconstitution, starting as early as 2 weeks after beginning of TP 5 treatment. High levels of circulating immature thymocytes and precursor T cells (defined by monoclonal antibodies OKT 6, OKT 9, and OKT 10) were present prior to therapy, and they steadily decreased during the first few weeks of study. During the same time, phenotypically mature T lymphocytes ( OKT 3 + and OKT 4 + OKT 8 + markedly increased, thereafter remaining at near normal levels. OKT 10 − cells appeared to rise again after 3 months of TP 5 treatment. In vitro function of T cells, assessed by PHA stimulation, and in vivo cell-mediated immunity (skin tests with Candida) were normal at 3 and at 2 months, respectively, after initiation of therapy. No severe infection episodes were recorded and normal development was achieved. No side effect or adverse reaction occurred. In these two patients the other features of the DiGeorge syndrome were successfully treated by early cardiac surgery and vitamin D therapy. The immunological reconstitution, in absence of functioning thymus observed in these two cases, provides further evidence of the effectiveness of long-term treatment with thymic hormones—with maintenance of the improvement of cell-mediated immunity.

Cytotoxic murine monoclonal antibody recognizing an ovine lymphocyte subpopulation similar to the human OKT4-positive set.

A cytotoxic murine immunoglobulin G2b monoclonal antibody was produced from immunization with ovine thymocytes. It reacts with a monomorphic determinant on ovine lymphocytes. This antibody 11.2 G11 does not react with B cells, lyses 50 to 60% of peripheral blood T cells, and precipitates a single chain protein with an apparent m.w. of 57,000. Its effect on mitogen- and antigen-driven lymphocyte proliferation supports its similarity in the sheep to the OKT4 antibody in humans.

Homogeneity of HLA-DQ,DR phenotypes in lymphocyte subsets of normal individuals

The study was performed to evaluate the association of HLA-DQ and DR antigens with the composition of peripheral blood lymphocytes. B cells, T cells, and T subsets were enumerated in 200 normal individuals by indirect immunofluorescence microscopy using the monoclonal antibodies OKT3, OKT4, OKT8, and OKIa1. Results were related to the HLA-DQ,DR phenotypes of the investigated population. No significant correlation between any of the lymphocyte subsets and of the HLA-DQ,DR antigens was found.

Exposure of K562 cells to anti-receptor monoclonal antibody OKT9 results in rapid redistribution and enhanced degradation of the transferrin receptor.

When the human erythroleukemia cell line K562 is treated with OKT9, a monoclonal antibody against the transferrin receptor, effects on receptor dynamics and degradation ensue. The apparent half-life of the receptor is decreased by greater than 50% as a result of OKT9 treatment. The transferrin receptor is also rapidly redistributed in response to OKT9 such that a lower percentage of the cellular receptors are displayed on the cell surface. OKT9 treatment also leads to a decrease in the total number of receptors participating in the transferrin cycle for cellular iron uptake. The reduction in iron uptake that results from the loss of receptors from the cycle leads to enhanced biosynthesis of the receptor. Receptors with bound OKT9 continue to participate in multiple cycles of iron uptake. However, OKT9 treatment appears to result in a relatively small increase per cycle in the departure of receptors from participation in iron uptake to a pathway leading to receptor degradation. Radiolabeled OKT9 is itself degraded by K562 cells and this degradation is inhibitable by leupeptin or chloroquine. In the presence of leupeptin, OKT9 treatment results in the enhanced intracellular accumulation of transferrin. Because the time involved in the transferrin cycle is shorter (12.5 min) than the normal half-life of the receptor (8 h), a small change in recycling efficiency caused by OKT9 treatment could account for the marked decrease in receptor half-life. In this paper the implications of these findings are discussed as they relate to systems in which receptor number is regulated by ligand.

An analysis of the cellular requirements for the production of soluble interleukin-2 receptors in vitro.

Following activation in vitro, peripheral blood mononuclear cells (PBMC) express cell-associated interleukin-2 receptors (IL-2R) and also release soluble IL-2R into culture supernatants. The present studies were undertaken to define which normal cells were responsible for the release of soluble IL-2R in vitro. Both cell-associated and soluble IL-2R were quantitatively measured with a "sandwich" enzyme-linked immunoassay employing two monoclonal antibodies. PBMC were separated into populations of surface immunoglobulin-negative cells (T cells and monocytes) and surface immunoglobulin-positive cells (B cells and monocytes), and the T-cell population was further separated into OKT4-positive (OKT4+) cells and OKT4-negative (OKT4-) cells. Following activation with phytohemagglutinin, pokeweed mitogen, and the monoclonal antibody OKT3, large amounts of soluble IL-2R were released by PBMC, unseparated T cells, OKT4+ T cells, and OKT4- T cells. The population containing B cells and monocytes made small but readily detectable amounts of soluble IL-2R when stimulated with these T-cell mitogens; likely the result of contaminating T cells in the population. However, when highly purified B cells were stimulated with Staphylococcus aureus Cowan and recombinant IL-2, they also released small amounts of soluble IL-2R. The release of soluble IL-2R by T cells appeared monocyte dependent when OKT3, but not phytohemagglutinin, was employed for activation, and monocytes themselves released no detectable IL-2R under the conditions employed. These studies define the cellular requirements for the release of soluble IL-2R in vitro and demonstrate that such receptors are released by B cells, T cells, and both OKT4+ and OKT4- T-cell subsets.

Expression of the transferrin receptor gene during the process of mononuclear phagocyte maturation.

The expression of transferrin receptors by blood monocytes, human alveolar macrophages, and in vitro matured macrophages was evaluated by immunofluorescence, radioligand binding, and Northern analysis, using the monoclonal anti-human transferrin receptor antibody OKT9, [125I]-labeled human transferrin and a [32P]-labeled human transferrin receptor cDNA probe, respectively. By immunofluorescence, the majority of alveolar macrophages expressed transferrin receptors (86 +/- 3%). The radioligand binding assay demonstrated the affinity constant (Ka) of the alveolar macrophage transferrin receptor was 4.4 +/- 0.7 X 10(8) M-1, and the number of receptors per cell was 4.4 +/- 1.2 X 10(4). In marked contrast, transferrin receptors were not present on the surface or in the cytoplasm of blood monocytes, the precursors of the alveolar macrophages. However, when monocytes were cultured in vitro and allowed to mature, greater than 80% expressed transferrin receptors by day 6, and the receptors could be detected by day 3. Consistent with these observations, a transferrin receptor mRNA with a molecular size of 4.9 kb was demonstrated in alveolar macrophages and in vitro matured macrophages but not in blood monocytes. Thus, although blood monocytes do not express the transferrin receptor gene, it is expressed by mature macrophages, an event that probably occurs relatively early in the process of monocyte differentiation to macrophages.

Enhancement of in vitro erythropoiesis by peripheral blood mononuclear cells from allogeneic marrow recipients in the early post-transplant period.

The effect of peripheral blood mononuclear cells (PBMCs) from bone marrow recipients on in vitro growth of erythroid burst-forming units (BFU-E) was studied. PBMCs were obtained from 5 allogeneic, 1 syngeneic and 1 autologous bone marrow recipient(s) at different intervals after transplantation. The number of BFU-E was significantly increased when donor bone marrow cells were co-cultured with PBMCs obtained from allogeneic marrow recipients in the early post-transplant period. No effect was observed using PBMCs obtained in the later post-transplant period, PBMCs from a syngeneic marrow recipient, or PBMCs from an autologous marrow recipient. The BFU-E enhancing activity was present among T cells and was abolished by treating them with OKT3 or OKT4 antibody and complement. These observations suggest that chimeric T lymphocytes, probably of the helper/inducer subset, from allogeneic marrow recipients in the early post-transplant period have a potent enhancing effect on in vitro erythropoiesis.

Phenotypic characterization of individual interferon-gamma-producing cells after OKT3 antibody activation.

This is the first study using a direct approach to determine the phenotype of interferon gamma (IFN-gamma)-producing cells after polyclonal T cell activation. Blood mononuclear cells (MNC) were cultured and stimulated by OKT3 antibody to produce IFN-gamma. They were then stained with a panel of monoclonal antibodies with specificity for cell surface antigens, fixed and permeabilized in suspension and stained for cytoplasmic IFN-gamma using monoclonal antibodies and indirect immunofluorescence technique. IFN-gamma appeared mainly as a local, polar accumulation in the cytoplasm in a juxtanuclear position, probably identical to the Golgi apparatus. After a culture period of 20 h, 6-11% and 0.2-0.6% of the OKT3-activated MNC from adults or newborns, respectively, produced IFN-gamma in cultures. Unstimulated cultures contained maximally 0.1% IFN-gamma-positive MNC. The majority of the IFN-gamma-producing MNC were T cells and expressed the T11 antigen and receptors for interleukin 2. To our surprise most of these T cells also carried receptors for complement 3bi (OKM1) but did not express class II molecules. Less than 50% displayed T4 or T8 antigens in three out of the four experiments performed. Natural killer cells, as recognized by Leu 7 or B73.1 antibodies, contributed only marginally to the IFN-gamma synthesis whereas B cells (OKB7) and monocytes (Leu M3) showed no production of IFN-gamma.

Stimulation and Expansion of a Human T-Cell Subpopulation by a Monoclonal Antibody to T-Cell Receptor Molecule

A murine monoclonal antibody (MAb) was obtained that showed unique specificity for the immunizing T-cell line HPB-ALL. This antibody, C37 (an IgG1,K) also reacted with a small (2-5%) population of normal peripheral blood T (PBL-T) cells. These C37-positive (C37+) cells were found in both the T4/Leu3+ and T8/Leu2+ subsets. Like OKT3 antibody, C37 induced T-cell mitogenesis with a peak proliferative response at day 3. In long-term cultures containing irradiated autologous feeder cells and IL-2, C37 antibody caused the selective expansion of C37+ T cells. On HPB-ALL cells C37 induced comodulation of the T3 molecule. C37 precipitated a disulfide-linked dimer characteristic of the T-cell antigen receptor consisting of an alpha-subunit (45-48 kD) and a beta-subunit (38-42 kD) from both C37+ T-cell blasts of a normal individual and HPB-ALL cells that were surface radioiodinated. However, the precipitated molecule isolated from C37 antibody-activated T-cell blasts exhibited a different pI from that isolated from HPB-ALL cells. Our studies indicate that C37 recognizes an epitope on the T-cell receptor molecule that is shared by a subpopulation of human T cells, which raises the possibility that multiple variable-region associated and/or framework-like determinants of the T-cell antigen receptor can be defined serologically and used in functional and molecular studies of T-cell subsets.

Activated lymphocytes in patients with newly diagnosed type 1 (insulin-dependent) diabetes mellitus.

The expression of activation antigens (transferrin receptor, IL-2 receptor and Ia antigen) on circulating T lymphocytes from Japanese children with Type 1 diabetes was studied using five monoclonal antibodies (Ab), OKT9, anti-Tac Ab, OKIa1, anti-human HLA-DR Ab and OKT3. For detecting Ia positive T cells, the dual staining technique using OKT3 and anti-Ia antibody was employed. Four out of six patients (67%) with newly diagnosed Type 1 diabetes showed a raised level of either OKT9 or Tac positive cells when examined at diagnosis. These patients, however, rapidly lost these activation antigens after the insulin therapy was started. In contrast, in 32 long-standing patients, only 2 (6%) had a high percentage of OKT9 positive cells and none of them demonstrated Tac positive cells. One out of six newly diagnosed patients or three out of 21 long-standing patients had a significantly high percentage of Ia-positive T cells compared with normal subjects. In poorly controlled long-standing patients whose HbA1 value was higher than 14%, none of them had an increased number of activated lymphocytes. Therefore, it is unlikely that insulin deficiency and hyperglycemia were responsible for the changes observed in these studies. Activated lymphocytes might be related to activation of the immune system involved in pathogenesis of Type 1 diabetes.

The role of transferrin in natural killer cell and IL-2-induced cytotoxic cell function

The growth factor transferrin (Tf) enhanced natural killer (NK) cell cytotoxicity. This enhancement was due to direct effects on NK cell function, and Tf treatment of the K562 target cell had no effect on their sensitivity. NK cells were highly enriched in the low-density large granular lymphocyte population (LGL) by Percoll gradient centrifugation. Despite the direct effect of Tf on NK cells, the number of cells expressing receptors for Tf (TfR) in NK-enriched LGL was the same as the NK-cell-depleted high-density small lymphocyte population (SL). All populations, tested without stimulation, had very few TfR + cells. Interleukin 2 (IL-2) could induce very high NK-like activity in the LGL but not in SL. Similarly, only LGL could be induced by IL-2 to express TfR. In serum-free cultures, only limited NK-like activity could be developed which was greatly enhanced by supplementing with Tf in the cultures. The importance of Tf in NK-like development was confirmed by modulating the expression of TfR in IL-2 containing cultures with mouse monoclonal antibody OKT9 specific for TfR. OKT9 totally abrogated the induction of cytotoxic activity by IL-2 against K562 and NK-resistant target. OKT9 inhibited the induction of cytotoxicity in both lymphocytes containing active NK cells and in those predepleted of active NK cells, indicating that the development of NK-like activity from both precursor populations requires Tf. The inhibition by OKT9 was only during the induction phase. The same antibody had no effect on the cytotoxicity of fresh NK cells or the mature IL-2-induced NK-like cells. Our data therefore do not support the hypothesis of TfR as the NK recognition structure. Instead, these results indicate that Tf is important for the development of NK and NK-like activities.

Lymphocyte subpopulations in solvent-exposed workers.

To estimate the cellular immune response of workers highly exposed to mixtures of organic solvents, subpopulations of peripheral blood lymphocytes (PBLs) were analyzed. For this, the PBLs of nine floorers (aged 25-58 years, exposure time 8-35 years) were subsequently labelled with monoclonal antibodies OKT4, OKT8, OKT11, anti-Leu 7 and anti-Leu 12. Analysis was made by a FACS IV cell sorter (Becton-Dickinson, USA). The control group consisted of matched pairs of healthy donors. In the exposed group we found a decrease in the OKT11 (all) T cell fraction, a decrease in the OKT4 helper cells, an increase in the anti-Leu 7 positive cells, mostly natural killer cells, an important increase in anti-Leu 12 labelled T cells, i.e., human B-lymphocytes, and no differences in the OKT8 suppressor cells. Total fluorescence intensity profiles between the exposed and the unexposed group did not differ, i.e., the marker density on the cell surfaces remained unchanged. Similar changes in lymphocyte subpopulations are found in states of immunodeficiency and immunogenetic forms of aplastic anemia, a disease whose etiological relationship may be due to long-term exposure to organic solvents.

Double fluorescence technique for measurement of complement-fixing antibody to lymphocyte subsets

A double fluorescent antibody method for quantitating human complement-fixing antibody to lymphocyte subclasses has been developed. The indicators in this system are a C6-deficient serum as a non-lytic source of complement, rhodamine-labeled anti-C3 and fluorescein-labeled murine monoclonal antibodies to human lymphocyte subsets. The basic procedure is to incubate lymphocytes with the unknown serum and then to add C6-deficient serum. The binding of C3 is indicated by staining with rhodamine-labeled anti-C3 and the subset class of the lymphocyte so stained is determined by binding of fluorescein-tagged anti-OKT4 or OKT8 antibodies. The occurrence of both redand green cell surface fluorescence denotes the presence of a complement-binding antibody to the lymphocyte subset defined by the monoclonal antibody. In addition to defining the specificity of complement-fixing anti-lymphocyte antibodies this technique is more sensitive than the microcytotoxicity assay.

Natural anti-bacterial activity against Salmonella typhi by human T4+ lymphocytes armed with IgA antibodies.

Peripheral blood mononuclear cells from normal volunteers possess natural anti-bacterial (NA) activity against S. typhi that can be measured in a 2-hr in vitro assay. Employing fractionation on nylon wool columns, Percoll gradients, plastic adherence, and E rosetting, the effector cell of NA activity appeared to be a lymphocyte of the T lineage rather than a macrophage, a B lymphocyte, or a large granular cell. Moreover, complement-dependent killing with monoclonal antibodies such as OKM1, OKB7, OKT8, 5.9 and the anti-natural killer cells AB8.28 did not reduce NA activity. On the contrary, this was completely inhibited when OKT3, OKT11, or OKT4 antibodies and complement were used to pretreat the effector lymphocytes. Indeed, T4+ cells sorted with a FACS displayed an extremely high NA activity against S. typhi. By pretreatment of peripheral lymphocytes with F(ab')2 fragments against human IgA, the NA activity was blocked. It is therefore suggested that NA activity by human cells might be a mechanism of defense against infections, acting as antibody-dependent cellular cytotoxicity expressed by T4+ lymphocytes coated with preexisting anti-Salmonella IgA antibodies.

Fucose-activated killer (FAK) cells: anomalous killers with augmented cytotoxic activity.

The effects of monosaccharides on various lymphocyte functions have provided useful probes for the study of cell-cell interactions. In this report, we show that a monosaccharide, alpha-L-fucose, significantly enhances the cytolytic capacity of MLC-induced or preincubated effector cells. The increase in activity was seen against cytotoxic T lymphocyte (CTL) targets (:relevant PHA blasts), natural killer cell (NK) targets (:K562), and natural cytotoxic cell (NC) targets (:MA-160). In addition, traditionally NK-insensitive targets (Raji cells, irrelevant and autologous PHA blasts) were lysed after preincubation of effector cells with fucose. Conversely, ADCC activity was not significantly increased with fucose induction. The addition of fucose directly to assay cultures did not enhance NK or CTL activity, whereas other sugars, such as alpha-methyl-D-mannoside and D-fructose, were inhibitory. The proportion of target-binding cells was not affected by preincubation with fucose, but the percentage of lytic conjugates was doubled. Significant augmentation of NK activity could be observed within 24 hr of incubation with alpha-L-fucose. Conversely, when fucose was added more than 24 hr after initiation of the culture, the increase in cytolytic activity was not observed. Parallel to the increase in cytolytic activity, after preincubation with alpha-L-fucose, an increase in the expression of a newly defined human NC cell marker, HNC-1A3, was observed. The HNC-1A3+ cells were not the major subpopulation responsible for fucose-induced activity, as ascertained by the use of positively sorted cells. The populations expressing antigens defined by the antibodies OKT8 and Leu-7 showed no quantitative change. The treatment of cells with OKM1 and complement (C) before culture eliminated fucose-enhanced killing, whereas similar treatment with OKT8 and C had no significant effect. The induction of fucose-activated killers (FAK) does not result in higher concentrations of interferon (IFN) in culture supernatants, in contrast to poly I:C, which induced both higher cytolytic activity and high titers of IFN. In addition, the induction of FAK was not sensitive to 100 ng/ml of cyclosporin A, suggesting that IL 2 did not play a major role in fucose activation of killing. These results provide strong evidence that alpha-L-fucose is capable of augmenting nonspecific activity by acting on OKM1+ precursors of cytotoxic cells and influencing a postbinding event.

Pattern of distribution of T lymphocytes, Langerhans cells and HLA‐DR bearing cells in normal human oral mucosa

The tissue distribution of helper/inducer and suppressor/cytotoxic T cells, Langerhans cells (LC) and HLA-DR bearing cells was determined in normal oral mucosa by use of monoclonal antibodies OKT4, OKT8, OKT6 and OKIa1, respectively. OKT4+ and OKT8+ cells were invariably present in normal oral epithelium and in the lamina propria. OKT8+ cells were consistently seen inside the basal cell layer of the epithelium. The distribution of LC in oral epithelium showed regional variation. In palatal epithelium LC were evenly distributed in the basal half of the epithelium, whereas in buccal mucosa the highest concentration of LC was seen in the epithelium overlying the tips of connective tissue papillae. OKIa1 stained dendritic cells in the epithelium and plump cells with small dendritic processes in the connective tissue. Some of the latter were located close to the basal cells of the epithelium. The consistent relationship between immunocompetent cells and the epithelium of the oral mucosa suggests the presence of a local immunologic defence barrier in the oral mucosa.

T cell unresponsiveness to the mitogenic activity of OKT3 antibody results from a deficiency of monocyte Fc gamma receptors for murine IgG2a and inability to cross-link the T3-Ti complex.

We recently identified defective monocyte accessory function as the cause of T cell unresponsiveness to the mitogenic activity of OKT3 antibody in cultures of peripheral blood mononuclear cells (PBMC) from five healthy subjects, members of one family. We now report that the underlying abnormality in nonresponders is at the level of monocyte Fc gamma receptors for murine IgG2a. T cell unresponsiveness was not restricted to the signal provided by OKT3 but occurred also for two other anti-T3 antibodies of the IgG2a subclass, in contrast to a normal proliferative response to IgG1 anti-T3 antibodies in one of the OKT3 nonresponders. By using cytofluorography, we found that monocytes from responders but not from nonresponders bound OKT3-FITC to their membrane. The binding could be blocked by mouse IgG2a and by human IgG, but not by mouse IgG1 nor by serum albumin. The data suggest that, through specific Fc gamma receptors for murine IgG2a, monocytes bind the Fc portion of OKT3 during T cell activation. The function of this Fc gamma receptor binding was further studied by culturing PBMC from nonresponders on plates coated with affinity-purified goat anti-mouse IgG antibodies as a substitute for monocyte Fc gamma receptors. The addition of OKT3 to nonresponder PBMC, cultured on such plates, resulted in T cell activation, as evidenced by thymidine incorporation, IL 2 production, and expression of IL 2 receptors. Soluble anti-mouse IgG was not able to substitute for monocyte Fc gamma receptors. The results demonstrate the existence of polymorphism in monocyte Fc gamma receptors for murine IgG2a. They also substantiate that an essential helper function of monocytes in T cell activation by anti-T3 is to provide a matrix for multimeric binding of the Fc portion of the anti-T3 antibodies in order to cross-link T3 molecules.

Demonstration of S-100 protein distribution in human lymphoid tissues by the avidin-biotin complex immunostaining method

Immunoreactivity for S-100 protein was investigated immunohistochemically in a series of 49 fixed and paraffin-embedded normal, reactive, and neoplastic human lymphoid tissue specimens. The avidin-biotin complex immunoperoxidase method was used, with overnight (12-hour) incubation with a commercially available antiserum to S-100 protein. In addition, cryostat sections were tested with DRC 1 monoclonal antibody to dendritic reticulum cells (DRCs) in three cases and with OKT6 antibody to interdigitating reticulum cells (IRCs) in nine cases. All tissues, including lymph nodes, tonsils, adenoid, spleens, appendices, thymuses, and tissues containing nodular reactive lymphoid infiltrates, demonstrated a consistent immune staining pattern. A striking network composed of dendritic processes that showed finely granular S-100 protein immunoreactivity was observed in most of the follicular germinal centers; a similar dendritic pattern was observed in the follicular centers when the corresponding frozen sections were immunostained with DRC 1. In the extrafollicular areas, the S-100-positive cells topographically and morphologically resembled the IRCs that were demonstrated by OKT6 antibody in the corresponding frozen sections. The results seem to indicate that cells topographically and morphologically similar to IRCs and DRCs in human lymphoid tissues from different sites share immunoreactivity for S-100 protein. The present study confirms the unexpected presence of S-100 protein in dendritic cells of follicular germinal centers by a simple and currently available method.